Methods of treatment using wisp polypeptides

ABSTRACT

The present invention relates to methods for the treatment and repair of cartilage, including cartilage damaged by injury or degenerative cartilagenous disorders, including arthritis, comprising the administration of WISP polypeptide. Optionally, the administration may be in combination with one or more cartilage agents (e.g., peptide growth factor, catabolism antagonist, osteo-, synovial, anti-inflammatory factor). Alternatively, the method provides for the treatment and repair of cartilage damaged by injury or degenerative cartilagenous disorders comprising the administration of WISP polypeptide in combination with standard surgical techniques. Alternatively, the method provides for the treatment and repair of cartilage damaged by injury or degenerative cartilagenous disorders comprising the administration of chondrocytes previously treated with an effective amount of WISP polypeptide.

RELATED APPLICATIONS

This application is a non-provisional application claiming priorityunder Section 119(e) to provisional application No. 60/241,222, filedOct. 16, 2000, the contents of which are incorporated herein byreference.

FIELD OF THE INVENTION

The present invention relates generally to methods of using WISPpolypeptides in the treatment of degenerative cartilagenous disordersand various immune related conditions.

BACKGROUND OF THE INVENTION

Connective tissue growth factor (CTGF) is a growth factor induced infibroblasts by many factors, including TGF-β, and is essential for theability of TGF-β to induce anchorage-independent growth (AIG), aproperty of transformed cells. CTGF was discovered in an attempt toidentify the type of platelet-derived growth factor (PDGF) dimerspresent in the growth media of cultured endothelial cells, and isrelated immunologically and biologically to PDGF. See U.S. Pat. No.5,408,040. CTGF also is mitogenic and chemotactic for cells, and hencegrowth factors in this family are believed to play a role in the normaldevelopment, growth, and repair of human tissue.

Seven proteins related to CTGF, including the chicken ortholog forCyr61, CEF10, human, mouse, and Xenopus laevis CTGF, and human, chicken,and Xenopus laevis Nov have been isolated, cloned, sequenced, andcharacterized as belonging to the CCN gene family. Oemar and Luescher,Arterioscler. Thromb. Vasc. Biol., 17: 1483-1489 (1997). The geneencoding Cyr61 has been found to promote angiogenesis, tumor growth, andvascularization. Babic et al., Proc. Natl. Acad. Sci. USA, 95: 6355-6360(1998). The nov gene is expressed in the kidney essentially at theembryonic stage, and alterations of nov expression, relative to thenormal kidney, have been detected in both avian nephroblastomas andhuman Wilms' tumors. Martinerie et al., Oncogene, 9: 2729-2732 (1994).Wt1 downregulates human nov expression, which downregulation mightrepresent a key element in normal and tumoral nephrogenesis. Martinerieet al., Oncogene, 12: 1479-1492 (1996). It has recently been proposedthat the CTGF, nov, and cyr61 genes, which encode secreted proteins thatcontain conserved sequences and IGFBP motifs in their N-termini and bindIGFs with low affinity, represent more members of the IGFBP superfamily,along with the low-affinity mac25/IGFBP-7 (Yamanaka et al., J. Biol.Chem., 272: 30729-30734 (1997)) and the high-affinity IGFBPs 1-6. CTGFunder this proposal would be designated IGFBP-8. Kim et al., Proc. Natl.Acad. Sci. USA, 94: 12981-12986 (1997).

The different members of the CCN family interact with various soluble ormatrix associated macromolecules in particular sulfated glycoconjugates(Holt et al., J. Biol. Chem., 265:2852-2855 (1990)). This interactionwas used to purify Cyr61 and CTGF by affinity chromatography onheparin-agarose (Frazier et al., J. Invest. Dermatol., 107:404-411(1996); Kireeva et al., Mol. Cell. Biol., 16:1326-1334 (1996)). Cyr61 issecreted and associated with both the extracellular matrix and the cellsurface due to its affinity for heparan sulfate (Yang et al., Cell.Growth Diff., 2:351-357 (1991)).

Recently, a protein was found in the mouse designated ELM1 that isexpressed in low metastatic cells. Hashimoto et al., J. Exp. Med., 187:289-296 (1998). The elm1 gene, a mouse homologue of WISP-1 disclosedbelow, is another member of the CTGF, Cyr61/Cef10, and neuroblastomaoverexpressed-gene family and suppresses in vivo tumor growth andmetastasis of K-1735 murine melanoma cells. Another recent article onrCop-1, the rat orthologue of WISP-2 described below describes the lossof expression of this gene after cell transformation. Zhang et al., Mol.Cell. Biol., 18:6131-6141 (1998).

CCN family members (with the exception of nov) are immediate earlygrowth-responsive genes that are thought to regulate cell proliferation,differentiation, embryogenesis, and wound healing. Sequence homologyamong members of the CCN gene family is somewhat high; however,functions of these proteins in vitro range from growth stimulatory(i.e., human CTGF) to growth inhibitory (i.e., chicken Nov and alsopossibly hCTGF). Further, some molecules homologous to CTGF areindicated to be useful in the prevention of desmoplasia, the formationof highly cellular, excessive connective tissue stroma associated withsome cancers, and fibrotic lesions associated with various skindisorders such as scleroderma, keloid, eosinophilic fasciitis, nodularfasciitis, and Dupuytren's contracture. Moreover, CTGF expression hasrecently been demonstrated in the fibrous stroma of mammary tumors,suggesting cancer stroma formation involves the induction of similarfibroproliferative growth factors as wound repair. Human CTGF is alsoexpressed at very high levels in advanced atherosclerotic lesions, butnot in normal arteries, suggesting it may play a role inatherosclerosis. Oemar and Luescher, supra.

Wnts are encoded by a large gene family whose members have been found inround worms, insects, cartilaginous fish, and vertebrates. Holland etal., Dev. Suppl., 125-133 (1994). Wnts are thought to function in avariety of developmental and physiological processes since many diversespecies have multiple conserved Wnt genes. McMahon, Trends Genet., 8:236-242 (1992); Nusse and Varmus, Cell, 69: 1073-1087 (1992). Wnt genesencode secreted glycoproteins that are thought to function as paracrineor autocrine signals active in several primitive cell types. McMahon,supra (1992); Nusse and Varmus, supra (1992). The Wnt growth factorfamily includes more than ten genes identified in the mouse (Wnt-1, -2,-3A, -3B, -4, -5A, -5B, -6, -7A, -7B, -8A, -8B, -10B, -11, -12, and -13)(see, e.g., Gavin et al., Genes Dev., 4: 2319-2332 (1990); Lee et al.,Proc. Natl. Acad. Sci. USA, 92: 2268-2272 (1995); Christiansen et al.,Mech. Dev., 51: 341-350 (1995)) and at least nine genes identified inthe human (Wnt-1, -2, -3, -5A, -7A, -7B, -8B, -10B, and -11) by cDNAcloning. See, e.g., Vant Veer et al., Mol. Cell. Biol., 4: 2532-2534(1984).

The Wnt-1 proto-oncogene (int-1) was originally identified from mammarytumors induced by mouse mammary tumor virus (MMTV) due to an insertionof viral DNA sequence. Nusse and Varmus, Cell, 31: 99-109 (1982). Inadult mice, the expression level of Wnt-1 mRNA is detected only in thetestis during later stages of sperm development. Wnt-1 protein is about42 KDa and contains an amino-terminal hydrophobic region, which mayfunction as a signal sequence for secretion (Nusse and Varmus, supra,1992). The expression of Wnt-2/irp is detected in mouse fetal and adulttissues and its distribution does not overlap with the expressionpattern for Wnt-1. Wnt-3 is associated with mouse mammary tumorigenesis.The expression of Wnt-3 in mouse embryos is detected in the neural tubesand in the limb buds. Wnt-5a transcripts are detected in the developingfore- and hind limbs at 9.5 through 14.5 days and highest levels areconcentrated in apical ectoderm at the distal tip of limbs. Nusse andVarmus, supra (1992). Recently, a Wnt growth factor, termed Wnt-x, wasdescribed (WO95/17416) along with the detection of Wnt-x expression inbone tissues and in bone-derived cells. Also described was the role ofWnt-x in the maintenance of mature osteoblasts and the use of the Wnt-xgrowth factor as a therapeutic agent or in the development of othertherapeutic agents to treat bone-related diseases.

Wnts may play a role in local cell signaling. Biochemical studies haveshown that much of the secreted Wnt protein can be found associated withthe cell surface or extracellular matrix rather than freely diffusiblein the medium. Papkoff and Schryver, Mol. Cell. Biol., 10: 2723-2730(1990); Bradley and Brown, EMBO J., 9: 1569-1575 (1990).

Studies of mutations in Wnt genes have indicated a role for Wnts ingrowth control and tissue patterning. In Drosophila, wingless (wg)encodes a Wnt-related gene (Rijsewik et al., Cell, 50: 649-657 (1987))and wg mutations alter the pattern of embryonic ectoderm, neurogenesis,and imaginal disc outgrowth. Morata and Lawerence, Dev. Biol., 56:227-240 (1977); Baker, Dev. Biol., 125: 96-108 (1988); Klingensmith andNusse, Dev. Biol., 166: 396-414 (1994). In Caenorhabditis elegans,lin-44 encodes a Wnt homolog which is required for asymmetric celldivisions. Herman and Horvitz, Development, 120: 1035-1047 (1994).Knock-out mutations in mice have shown Wnts to be essential for braindevelopment (McMahon and Bradley, Cell, 62: 1073-1085 (1990); Thomas andCappechi, Nature, 346: 847-850 (1990)), and the outgrowth of embryonicprimordia for kidney (Stark et al., Nature, 372: 679-683 (1994)), tailbud (Takada et al., Genes Dev., 8: 174-189 (1994)), and limb bud. Parrand McMahon, Nature, 374: 350-353 (1995). Overexpression of Wnts in themammary gland can result in mammary hyperplasia (McMahon, supra (1992);Nusse and Varmus, supra (1992)), and precocious alveolar development.Bradbury et al., Dev. Biol., 170: 553-563 (1995).

Wnt-5a and Wnt-5b are expressed in the posterior and lateral mesodermand the extraembryonic mesoderm of the day 7-8 murine embryo. Gavin etal., supra (1990). These embryonic domains contribute to the AGM regionand yolk sac tissues from which multipotent hematopoietic precursors andHSCs are derived. Dzierzak and Medvinsky, Trends Genet., 11: 359-366(1995); Zon et al., in Gluckman and Coulombel, ed., Colloque, INSERM,235: 17-22 (1995), presented at the Joint International Workshop onFoetal and Neonatal Hematopoiesis and Mechanism of Bone Marrow Failure,Paris France, Apr. 3-6, 1995; Kanatsu and Nishikawa, Development, 122:823-830 (1996). Wnt-5a, Wnt-10b, and other Wnts have been detected inlimb buds, indicating possible roles in the development and patterningof the early bone microenvironment as shown for Wnt-7b. Gavin et al.,supra (1990); Christiansen et al., Mech. Devel., 51: 341-350 (1995);Parr and McMahon, supra (1995).

The Wnt/Wg signal transduction pathway plays an important role in thebiological development of the organism and has been implicated inseveral human cancers. This pathway also includes the tumor suppressorgene, APC. Mutations in the APC gene are associated with the developmentof sporadic and inherited forms of human colorectal cancer. The Wnt/Wgsignal leads to the accumulation of beta-catenin/Armadillo in the cell,resulting in the formation of a bipartite transcription complexconsisting of beta-catenin and a member of the lymphoid enhancer bindingfactor/T cell factor (LEF/TCF)HMG box transcription factor family. Thiscomplex translocates to the nucleus where it can activate expression ofgenes downstream of the Wnt/Wg signal, such as the engrailed andUltrabithorax genes in Drosophila.

For a review on Wnt, see Cadigan and Nusse, Genes & Dev., 11: 3286-3305(1997).

Pennica et al., Proc. Natl. Acad. Sci., 95:14717-14722 (1998) describethe cloning and characterization of two genes, WISP-1 and WISP-2, thatare up-regulated in the mouse mammary epithelial cell line C57MGtransformed by Wnt-1, and a third related gene, WISP-3. Pennica et al.report that these WISP genes may be downstream of Wnt-1 signaling andthat aberrant levels of WISP expression in colon cancer may play a rolein colon tumorigenesis. WISP-1 has recently been identified as aβ-catenin-regulated gene and the characterization of its oncogenicactivity demonstrated that WISP-1 might contribute to β-catenin-mediatedtumorigenesis (Xu et al., Gene & Develop., 14:585-595 (2000)). WISP-1overexpression in normal rat kidney cells (NRK-49F) inducedmorphological transformation, accelerated cell growth and enhancedsaturation density. In addition, these cells readily form tumors wheninjected into nude mice suggesting that WISP-1 may play some role intumorigenesis (Xu et al., supra 2000).

Hurvitz et al., Nature Genetics, 23:94-97 (1999) describe a studyinvolving WISP3 in which nine different mutations of WISP3 in unrelatedindividuals were found to be associated with the autosomal recessiveskeletal disorder, progressive pseudorheumatoid dysplasia (PPD). WISP3expression by RT-PCR was observed by Hurvitz et al. in humansynoviocytes, articular cartilage chondrocytes, and bone-marrow-derivedmesenchymal progenitor cells.

PCT application WO98/21236 published May 22, 1998 discloses a humanconnective tissue growth factor gene-3 (CTGF-3) encoding a 26 kD memberof the growth factor superfamily. WO98/21236 discloses that the CTGF-3amino acid sequence was deduced from a human osteoblast cDNA clone, andthat CTGF-3 was expressed in multiple tissues like ovary, testis, heart,lung, skeletal muscle, adrenal medulla, adrenal cortex, thymus,prostate, small intestine and colon.

Several investigators have documented changes in the proteoglycancomposition in neoplasms. Especially, a marked production of chondroitinsulfate proteoglycan is a well recognized phenomenon in a variety ofmalignant tumors. In addition, the expression of decorin, a dermatansulfate containing proteoglycan, has been shown to be well correlatedwith malignancy in human carcinoma (Adany et al., J. Biol. Chem.,265:11389-11396 (1990); Hunzlemann et al., J. Invest. Dermatol.,104:509-513 (1995)). Recently, it was demonstrated that decorinsuppresses the growth of several carcinomas (Santra 1997). Although thefunction of decorin in tumorigenic development is not fully understood,it was proposed that the decorin expression in the peritumorous stromamay reflect a regional response of the host connective tissue cells tothe invading neoplastic cells (Stander et al., Gene Therapy, 5:1187-1194(1999)).

For a recent review of various members of the connective tissue growthfactor/cysteine-rich 61/nephroblastoma overexpressed (CNN) family, andtheir respective properties and activities, see Brigstock, EndocrineReviews, 20:189-206 (1999).

Degenerative cartilagenous disorders broadly describe a collection ofdiseases characterized by degeneration or metabolic abnormalities of theconnective tissues which can be manifested by pain, stiffness andlimitation of motion of the affected body parts. The origin of thesedisorders can be, for example, pathological or as a result of trauma orinjury.

Osteoarthritis (OA), also known as osteoarthrosis or degenerative jointdisease, is typically the result of a series of localized degenerativeprocesses that affect the articular structure and result in pain anddiminished function. OA is often accompanied by a local inflammatorycomponent that may accelerate joint destruction. OA is characterized bydisruption of the smooth articulating surface of cartilage, with earlyloss of proteoglycans (PG) and collagens, followed by formation ofclefts and fibrillation, and ultimately by full-thickness loss ofcartilage. OA symptoms include local pain at the affected joints,especially after use. With disease progression, symptoms may progress toa continuous aching sensation, local discomfort and cosmetic alterationssuch as deformity of the affected joint.

In contrast to the localized nature of OA, rheumatoid arthritis (RA) isa systemic, inflammatory disease which likely begins in the synovium,the tissues surrounding the joint space. RA is a chronic autoimmunedisorder characterized by symmetrical synovitis of the joint andtypically affects small and large diarthrodial joints, leading to theirprogressive destruction. As the disease progresses, the symptoms of RAmay also include fever, weight loss, thinning of the skin, multiorganinvolvement, scleritis, corneal ulcers, formation of subcutaneous orsubperiosteal nodules and premature death. While the cause(s) or originsof RA and OA are distinctly different, the cytokines and enzymesinvolved in cartilage destruction appear to be similar.

Peptide growth factors are believed to be important regulators ofcartilage growth and cartilage cell (chondrocyte) behavior (i.e.,differentiation, migration, division, and matrix synthesis or breakdown)F. S. Chen et al., Am J. Orthop. 26: 396-406 (1997). Growth factors thathave been previously proposed to stimulate cartilage repair includeinsulin-like growth factor (IGF-1), Osborn, J. Orthop. Res. 7: 35-42(1989); Florini & Roberts, J. Gerontol. 35: 23-30 (1980); basicfibroblast growth factor (bFGF), Toolan et al., J. Biomec. Mat. Res. 41:244-50 (1998); Sah et al., Arch. Biochem. Biophys. 308: 137-47 (1994);bone morphogenetic protein (BMP), Sato & Urist, Clin. Orthop. Relat.Res. 183: 180-87 (1984); Chin et al., Arthritis Rheum. 34: 314-24 (1991)and transforming growth factor beta (TGF-beta), Hill & Logan, Prog.Growth Fac. Res. 4: 45-68 (1992); Guerne et al., J. Cell Physiol. 158:476-84 (1994); Van der Kraan et al., Ann. Rheum. Dis. 51: 643-47 (1992).

Insulin-like growth factor (IGF-1) stimulates both matrix synthesis andcell proliferation in culture, K. Osborn. J. Orthop. Res. 7: 35-42(1989), and insufficiency of IGF-1 may have an etiologic role in thedevelopment of osteoarthritis. R. D. Coutts, et al., InstructionalCourse Lect. 47: 487-94, Amer. Acad. Orthop. Surg. Rosemont, Ill.(1997). Some studies indicate that serum IGF-1 concentrations are lowerin osteoarthritic patients than control groups, while other studies havefound no difference. Nevertheless, both serum IGF-1 levels andchondrocyte responsiveness to IGF-1 decrease with age. J. R. Florini &S. B. Roberts, J. Gerontol. 35: 23-30 (1980). Thus, both the decreasedavailability of IGF-1 as well as diminished chondrocyte responsivenessto IGF-1 may contribute to cartilage homeostasis and lead todegeneration with advancing age.

IGF-1 has been proposed for the treatment of prevention ofosteoarthritis. Intra-articular administration of IGF-1 in combinationwith sodium pentosan polysulfate (a chondrocyte catabolic activityinhibitor) caused improved histological appearance, and near-normallevels of degradative enzymes (neutral metalloproteinases andcollagenase), tissue inhibitors of metalloproteinase and matrixcollagen. R. A. Rogachefsky, et al., Ann. NY Acad. Sci. 732: 889-95(1994). The use of IGF-1 either alone or as an adjuvant with othergrowth factors to stimulate cartilage regeneration has been described inWO 91/19510, WO 92/13565, U.S. Pat. No. 5,444,047, and EP 434,652,

Bone morphogenetic proteins (BMPs) are members of the large transforminggrowth factor beta (TGF-β) family of growth factors. In vitro and invivo studies have shown that BMP induces the differentiation ofmesenchymal cells into chondrocytes. K. Sato & M. Urist, Clin. Orthop.Relat. Res. 183: 180-87 (1984). Furthermore, skeletal growth factor andcartilage-derived growth factors have synergistic effects with BMP, asthe combination of these growth factors with BMP and growth hormoneinitiates mesenchymal cell differentiation. Subsequent proliferation ofthe differentiated cells are stimulated by other factors. D. J. Hill & ALogan, Prog. Growth Fac. Res. 4: 45-68 (1992).

Transforming growth factor beta (TGF-β) is produced by osteoblasts,chondrocytes, platelets, activated lymphocytes, and other cells. R. D.Coutts et al., supra. TGF-β can have both stimulatory and inhibitoryproperties on matrix synthesis and cell proliferation depending on thetarget cell, dosage, and cell culture conditions. P. Guerne et al., J.Cell Physiol. 158: 476-84 (1994); H. Van Beuningen et al., Ann. Rheum.Dis. 52: 185-91 (1993); P. Van der Kraan et al., Ann. Rheum. Dis. 51:643-47 (1992). Furthermore, as with IGF-1, TGF-β responsiveness isdecreased with age. P. Guerne et al., J. Cell Physiol. 158: 476-84(1994). However, TGF-β is a more potent stimulator of chondrocyteproliferation than other growth factors, including platelet-derivedgrowth factor (PDGF), bFGF, and IGF-1 (Guerne et al., supra), and canstimulate proteoglycan production by chondrocytes. TGF-β alsodown-regulates the effects of cytokines which stimulate chondrocytecatabolism Van der Kraan et al., supra. In vivo, TGF-β inducesproliferation and differentiation of mesenchymal cells into chondrocytesand enhances repair of partial-thickness defects in rabbit articularcartilage. E. B. Hunziker & L. Rosenberg, Trans. Orthopaed. Res. Soc.19: 236 (1994).

While some investigators have focused on the use of certain growthfactors to repair cartilage or chondrocyte tissue, others have looked atinhibiting the activity of molecules which induce cartilage destructionand/or inhibit matrix synthesis. One such molecule is the cytokineIL-1alpha, which has detrimental effects on several tissues within thejoint, including generation of synovial inflammation and up-regulationmatrix metalloproteinases and prostaglandin expression. V. Baragi, etal., J. Clin. Invest. 96: 2454-60 (1995); V. M. Baragi et al.,Osteoarthritis Cartilage 5: 275-82 (1997); C. H. Evans et al., J.Keukoc. Biol. 64: 55-61 (1998); C. H Evans and P. D. Robbins, J.Rheumatol. 24: 2061-63 (1997); R. Kang et al., Biochem. Soc. Trans. 25:533-37 (1997); R. Kang et al., Osteoarthritis Cartilage 5: 139-43(1997). One means of antagonizing IL-1alpha is through treatment withsoluble IL-1 receptor antagonist (IL-1ra), a naturally occurring proteinthat prevents IL-1 from binding to its receptor, thereby inhibiting bothdirect and indirect effects of IL-1 on cartilage. In mammals only oneprotease, named interleukin 1beta-convertase (ICE), can specificallygenerate mature, active IL-1alpha. Inhibition of ICE has been shown toblock IL-1alpha production and may slow arthritic degeneration (reviewedin Martel-Pelletier J. et al. Front. Biosci. 4: d694-703). The solubleIL-1 receptor antagonist (IL-1ra), a naturally occurring protein thatcan inhibit the effects of IL-1 by preventing IL-1 from interacting withchondrocytes, has also been shown to be effective in animal models ofarthritis and is currently being tested in humans for its ability toprevent incidence or progression of arthritis. Other cytokines, such asIL-1beta, tumor necrosis factor-alpha, interferon gamma, IL-6, and IL-8have been linked to increased activation of synovial fibroblast-likecells, chondrocytes and/or macrophages. The inhibition of thesecytokines may be of therapeutic benefit in preventing inflammation andcartilage destruction. Molecules which inhibit TNF-alpha activity havebeen shown to have beneficial effects on the joints of patients withrheumatoid arthritis.

Cartilage matrix degradation is believed to be due to cleavage of matrixmolecules (proteoglycans and collagens) by proteases (reviewed inWoessner J F Jr., “Proteases of the extracellular matrix”, in Mow, V.,Ratcliffe, A. (eds): Structure and Function of Articular Cartilage. BocaRaton, Fla., CRC Press, 1994 and Smith R. L., Front. In Biosci.4:d704-712. While the key enzymes involved in matrix breakdown have notyet been clearly identified, matrix metalloproteinases (MMPs) and“aggrecanases” appear to play key roles in joint destruction. Inaddition, members of the serine and cysteine family of proteinases (forexample, the cathepsins and urokinase or tissue plasminogen activator(uPA and tPA)) may also be involved. Plasmin, urokinase plasminogenactivator (uPA) and tissue plasminogen activator (tPA) may play animportant role in the activation pathway of the metalloproteinases.Evidence connects the closely related group of cathepsin B, L and S tomatrix breakdown, and these cathepsins are somewhat increased in OA.Many cytokines, including IL-1, TNF-alpha and LIF induce MMP expressionin chondrocytes. Induction of MMPs can be antagonized by TGF-β and IL-4and potentiated, at least in rabbits, by FGF and PDGF. As shown byanimal studies, inhibitors of these proteases (MMPs and aggrecanases)may at least partially protect joint tissue from damage in vivo.

Nitric oxide (NO) may also play a substantial role in the destruction ofcartilage. Ashok et al., Curr. Opin. Rheum. 10: 263-268 (1998). Unlikenormal cartilage which does not produce NO unless stimulated withcytokines such as IL-1, cartilage obtained from osteoarthritic jointsproduces large amounts of nitric oxide for over 3 days in culturedespite the absence of added stimuli. Moreover, inhibition of NOproduction has been shown to prevent IL-1 mediated cartilage destructionand chondrocyte death as well as progression of osteoarthritis in animalmodels.

SUMMARY OF THE INVENTION

Applicants have surprisingly found that WISP polypeptides have usefulactivities, such as the ability to stimulate or enhance chondrocytedifferentiation or proliferation, and thus, WISP polypeptides can beuseful for the treatment, repair or protection of cartilage, includingcartilage damaged as a result of a cartilagenous disorder and/or injury.

In one embodiment, the present invention concerns a method for thetreatment of cartilage damaged as a result of a cartilagenous disordercomprising contacting said cartilage with an effective amount of WISPpolypeptide. WISP polypeptides contemplated for use in the inventioninclude but are not limited to WISP-1, WISP-2 and WISP-3 polypeptidesand variants thereof, described further below. Optionally, the cartilageis articular cartilage, and the amount of WISP polypeptide employed is atherapeutically effective amount. In a preferred embodiment, thecartilagenous disorder is osteoarthritis or rheumatoid arthritis. Themethods may be conducted in vivo, such as by administering thetherapeutically effective amount of WISP polypeptide to the mammal, orex vivo, by contacting said cartilage tissue with an effective amount ofWISP polypeptide in culture and then transplanting the treated cartilagetissue into the mammal. In addition, the methods may be conducted byemploying WISP polypeptide alone as a therapeutic agent, or incombination with an effective amount of a cartilage agent or othertherapeutic technique. For example, the WISP polypeptide may be employedin combination with any standard cartilage surgical technique. The WISPpolypeptide may be administered prior, after and/or simultaneous to thestandard cartilage surgical technique.

In a further embodiment, the present invention concerns a method for thetreatment of cartilage damaged by injury or preventing the initial orcontinued damage comprising contacting said cartilage with an effectiveamount of WISP polypeptide. More specifically, the injury treated ismicrodamage or blunt trauma, a chondral fracture, an osteochondralfracture, or damage to tendons, menisci, or ligaments. In a specificaspect, the injury can be the result of excessive mechanical stress orother biomechanical instability resulting from a sports injury orobesity. Alternatively, the present invention concerns a method oftreating or facilitating the repair of bone fractures comprisingcontacting the region of the bone injury with an effective amount ofWISP polypeptide.

In another embodiment, the invention concerns a method of enhancing,stimulating or promoting the differentiation of chondrocytes orchondrocyte precursor cells by contacting the chondrocytes orchondrocyte precursor cells with an effective amount of WISPpolypeptide.

In another embodiment, the present invention concerns a kit or articleof manufacture, comprising WISP polypeptide and a carrier, excipientand/or stabilizer (e.g. a buffer) in suitable packaging. The kit orarticle preferably contains instructions for using WISP polypeptide totreat cartilage damaged or to prevent initial or continued damage tocartilage as a result of a cartilagenous disorder. Alternatively, thekit may contain instructions for using WISP polypeptide to treat acartilagenous disorder.

More particular embodiments of the present invention include methods oftreating mammalian cartilage cells or tissue, comprising contactingmammalian cartilage cells or tissue damaged from a degenerativecartilagenous disorder (or damaged from an injury) with an effectiveamount of WISP polypeptide, wherein said WISP polypeptide is apolypeptide selected from the group consisting of:

-   a) a WISP-1 polypeptide comprising amino acids 23 to 367 of SEQ ID    NO:3;-   b) a WISP-1 polypeptide comprising amino acids 1 to 367 of SEQ ID    NO:3;-   c) a WISP-1 polypeptide having at least 90% identity to the    polypeptide of a) or b);-   d) a biologically active fragment of the polypeptide of a) or b);-   e) a WISP-2 polypeptide comprising amino acids 24 to 250 of SEQ ID    NO:10;-   f) a WISP-2 polypeptide comprising amino acids 1 to 250 of SEQ ID    NO:10;-   g) a WISP-2 polypeptide having at least 90% identity to the    polypeptide of e) of f);-   h) a biologically active fragment of the polypeptide of e) or f);-   i) a WISP-3 polypeptide comprising amino acids 34 to 372 of SEQ ID    NO:9;-   j) a WISP-3 polypeptide comprising amino acids 1 to 372 of SEQ ID    NO:9;-   k) a WISP-3 polypeptide comprising amino acids 16 to 354 of SEQ ID    NO:8;-   l) a WISP-3 polypeptide comprising amino acids 1 to 354 of SEQ ID    NO:8;-   m) a WISP-3 polypeptide having at least 90% identity to the    polypeptide of i), j), k) or l); and    a biologically active fragment of the polypeptide of i), j), k) or    l). Optionally, the WISP-1 polypeptide has at least 90% identity to    the polypeptide of a) or b), wherein said polypeptide WISP-1    stimulates chondrocyte proliferation or differentiation.    Alternatively, the WISP-1 polypeptide is a biologically active    fragment of the WISP-1 polypeptide of a) or b), wherein said    biologically active fragment stimulates chondrocyte proliferation or    differentiation. Optionally, the WISP-2 polypeptide has at least 90%    identity to the polypeptide of e) or f), wherein said polypeptide    WISP-2 stimulates chondrocyte proliferation or differentiation.    Alternatively, the WISP-2 polypeptide is a biologically active    fragment of the WISP-2 polypeptide of e) or f), wherein said    biologically active fragment stimulates chondrocyte proliferation or    differentiation. Optionally, the WISP-3 polypeptide has at least 90%    identity to the polypeptide of i), j), k) or l), wherein said    polypeptide WISP-3 stimulates chondrocyte proliferation or    differentiation. Alternatively, the WISP-3 polypeptide is a    biologically active fragment of the WISP-3 polypeptide of i), j), k)    or l), wherein said biologically active fragment stimulates    chondrocyte proliferation or differentiation. The WISP polypeptides    referred to above may be linked to one or more polyethylene glycol    molecules. Optionally, the WISP polypeptides may be linked to an    epitope tag or immunoglobulin molecule. In the methods, the    cartilage may be articular cartilage, and the degenerative    cartilagenous disorder may be rheumatoid arthritis or    osteoarthritis.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1G show the binding of WISP-1 to different cell lines. Cellswere seeded in chamber slides and cultured overnight. The next day, thenon-specific binding sites were blocked and the cells were incubatedwith 1 nM of mWISP-1-IgG (1A and 1B) or without mWISP-1-IgG (1C) for 1hour. The cells were washed, fixed and the binding of WISP-1-IgG wasdetected by immunofluorescence using a biotinylated anti-human IgGantibody and the indirect tyramide substrate amplification procedurefollowed with FITC conjugated streptavidin. In 1A, there are grouped thecell lines to which mWISP-1-IgG bound. The picture represents thetypical fluorescent signal found on a surface of NRK cells followingmWISP-1-IgG binding. In 1B, there are grouped the cell lines to whichmWISP-1-IgG did not bind. The picture represents the typical fluorescentsignal found on surfaces of RAG cells following mWISP-1-IgG binding. Thepicture in 1C represents the typical fluorescent signal found onsurfaces of NRK cells when mWISP-1-IgG was omitted from the bindingprocedure. Slide mounted human colon tumor sections were brought to roomtemperature and washed, saturated and incubated for 1 hour in HBS-C/3%BSA and 1 nM WISP-1-Fc (1D and 1E). In parallel, the immunofluorescentdetection of vimentin was performed on adjacent sections as described inExample 1 (1F and 1G).

FIGS. 2A-2B show the binding of mWISP-1-IgG to human skin fibroblastsconditioned media. Serum free conditioned media of human skinfibroblasts was prepared as described in the section “Purification ofWISP-1 Binding Factors”. Fifty microliters of conditioned media wascoated in duplicate in microtitration wells. The non-specific bindingsites were saturated by incubation with HBS-C containing 3% BSA and thewells were incubated for 2 hours with mWISP-1-IgG. The wells were washedand incubated for 1 hour with horseradish peroxidase conjugatedanti-human IgG Fc′. After 6 washes with HBS-C containing 0.3% BSA, thesignal was visualized using a horseradish peroxidase chromogenicsubstrate. The reaction was stopped with 1 M phosphoric acid and the ODat 450 nm was measured. 2A shows binding of 1 nM of mWISP-1-IgG to wellscoated with serial dilutions of conditioned media; 2B shows binding ofserial dilutions of mWISP-1-IgG to wells coated with 0.5 μl of humanskin fibroblast conditioned media.

FIGS. 3A-3B show the binding of mWISP-1-IgG to a chondroitinase Bsensitive factor of human skin fibroblast conditioned media. In 3A,fifty microliters of conditioned media was coated in duplicate inmicrotitration wells and the non-specific binding sites were saturatedby incubation with HBS-C containing 3% BSA. One nanomolar WISP-1-IgG wasincubated for 2 hours in the absence or the presence of 1 M NaCl, 100 mMEDTA or 0.05% Tween-20. The wells were washed and incubated for 1 hourwith horseradish peroxidase conjugated anti-human IgG Fc′. After 6washes with HBS-C containing 0.3% BSA, the signal was visualized using ahorseradish peroxidase chromogenic substrate. The reaction was stoppedwith 1 M phosphoric acid and the OD at 450 nm was measured. In 3B, 50 μlof HBS-C containing 0.5 U/ml chondroitinase ABC (Ch ABC), 0.5 U/mlchondroitinase AC II (Ch AC II), 0.5 U/ml chondroitinase B (Ch B), 0.5U/ml chondroitinase C (Ch C), 0.5 U/ml chondroitin-4-sulfatase(Ch-4-Sulf), 0.5 U/ml chondroitin-6-sulfatase (Ch-6-Sulf), 0.5 U/mlheparinase (Hep), 0.5 U/ml hyaluronidase (Hyal), 0.5 U/ml neuramimidase(Neuram) or 100 μg/ml proteinase K (Prot K) were added to the coatedwells and incubated for 2 hours at 37° C. The wells were washedextensively, the non specific binding sites were saturated and 1 nMmWISP-1-IgG was incubated for 2 hours at room temperature. The wellswere washed and binding of WISP-1-IgG was measured.

FIGS. 4A-4B show purification of WISP-1 binding factors from human skinfibroblast conditioned media. In 4A, the serum free conditioned mediafrom human skin fibroblasts was collected after three days of culture,concentrated, transferred to a buffer containing 20 mM Tris-HCl pH 7.4and 300 mM NaCl, and applied on a Q-Sepharose anion exchangechromatography column. The column was washed and the retained proteinswere desorbed with an increasing concentration of NaCl. The presence ofa WISP-1 binding factor was analyzed in each fraction using a solidphase binding assay. In 4B, fraction 15 (indicated by a * in FIG. 4A)was incubated at 37° C. for 2 hours in the presence (+) or the absence(−) of 0.1 U of chondroitinase ABC. The samples were separated bySDS-PAGE under reducing conditions and the gels were silver stained. Theindicated bands were identified by mass spectroscopy.

FIGS. 5A-5B show WISP-1 binding to decorin and biglycan. In 5A,microtiter wells were coated with serial dilutions of decorin (filledcircles) or biglycan (empty circles). Non-specific binding sites weresaturated and 0.25 nM of mWISP-1-IgG was incubated for 2 hours. Thewells were washed and incubated with horseradish peroxidase conjugatedanti-human IgG Fc′ (2 μg/ml) for 1 hour. After 6 washes with PBScontaining 0.05% Tween-20, a signal was developed by the incubation of achromogenic substrate. The color development was stopped by the additionof 1 M phosphoric acid and the O.D. at 450 nm was measured. In 5B, fiftymilliliters of human skin fibroblast conditioned media were coated inwells of microtiter plates. Non-specific binding sites were saturatedand 0.25 nM of WISP-1-IgG was incubated in the presence of variousconcentrations of decorin (filled circles) or biglycan (empty circles)for 2 hours. The binding of mWISP-1-IgG was evaluated as described in5A.

FIG. 6 shows mWISP-1-IgG binding to glycosaminoglycans. Serum freeconditioned media of human skin fibroblasts was prepared as describedbelow in the Examples. Fifty μl of conditioned media were coated inwells of microplates overnight at 4° C., the non specific binding siteswere saturated and the wells were incubated for 2 hours at roomtemperature with 0.5 nM of WISP-1-IgG in the presence of variousconcentrations of different glycosaminoglycans. The wells were washed, asignal was developed using a chromogenic substrate and the O.D. at 450nm was measured. Chondroitin sulfate A (filled circles); dermatansulfate (empty circles); chondroitin sulfate C (filled triangles);chondroitin sulfate D (empty triangles); chondroitin sulfate E (filledsquares); heparin (X); heparan sulfate (empty squares).

FIGS. 7A-7I show that WISP-1 binding to human skin fibroblasts iscompeted by dermatan sulfate. Human skin fibroblasts were seeded inchamber slides. The non specific binding sites were saturated and 1 nMWISP-1-IgG was incubated for 1 hour at room temperature in the absence(7A) or the presence (7B) of 50 μg/ml chondroitin sulfate A (“CSA”),dermatan sulfate (“DS”); (7C), chondroitin sulfate C (“CS C”); (7D),chondroitin sulfate D (“CS D”); (7E), chondroitin sulfate E (“CS E”)(7F); heparin (“Hep”) (7G) or heparan sulfate (“HS”) (7H). The cellswere washed and fixed and the binding of WISP-1-IgG was detected byimmunofluorescence using a biotinylated anti-human IgG antibody and theindirect tyramide substrate amplification procedure ended with FITCconjugated streptavidin. The relative fluorescence intensity of acquireddigital images was measured by morphometric analysis (7I).

FIGS. 8A-8G show that WISP-1 binding to human skin fibroblasts isabolished by the digestion of the cell surface with chondroitinase B.Human skin fibroblasts were incubated for 2 hours at 37° C. in theabsence (8A), or the presence of 0.1 U of chondroitinase ABC (Ch ABC);(8B), chondroitinase B (“Ch B”); (8C), chondroitinase C (“Ch C”); (8D),heparinase (“Hep”) (8E). The cells were washed, the non specific bindingsites were saturated and 1 nM WISP-1-IgG was incubated for 1 hour atroom temperature. After 3 washes, the cells were fixed and the bindingof WISP-1-IgG was detected by immunofluorescence using a biotinylatedanti-human IgG antibody and the indirect tyramide substrateamplification procedure ended with FITC conjugated streptavidin. FIG. 8Frepresents a negative control in which undigested cells were used butmWISP-1-IgG was omitted from the binding procedure. The relativefluorescence intensity of acquired digital images was measured bymorphometric analysis (8G).

FIGS. 9A-9D show that WISP-1 binding to human skin fibroblasts iscompeted by decorin and biglycan. Human skin fibroblasts were seeded inchamber slides and the non specific binding sites were saturated. Onenanomolar mWISP-1-IgG was incubated for 1 hour at room temperature inthe presence of 1 mg/ml decorin (9A) or biglycan (9B), or in the absenceof added competitors (9C). The cells were washed and fixed and thebinding of WISP-1-IgG was detected by immunofluorescence using abiotinylated anti-human IgG antibody and the indirect tyramide substrateamplification procedure ended with FITC conjugated streptavidin. Therelative fluorescence intensity of acquired digital images was measuredby morphometric analysis (9D).

FIG. 10 shows the adhesion of different mutants of CHO cells to WISP-1.Cells were taken up in PBS containing 2 mM EDTA and then washed andresuspended in serum free Ham-F12/LGDMEM (50:50) containing 1% BSA. Cellsuspension was added to microtiter wells coated with WISP-1 andincubated at 37° C. for 2 hours. The wells were washed 3× with PBS, thesupernatant removed and the number of adherent cells measured usingCyQUANT from Molecular Probes. Adhesion of CHO-K1 cells to microtiterwells coated with WISP-1 was used as 100% and all values were correctedfor nonspecific adhesion to microtiter wells coated with BSA.

FIG. 11 shows the adhesion of human skin fibroblasts to WISP-1. Cellswere taken up in PBS containing 15 mM EDTA and then washed andresuspended in serum free Ham-F12/LGDMEM (50:50) containing 1% BSA. Cellsuspension was added to microtiter wells in the absence or the presenceof 100 μg/ml of dermatan sulfate (i.e., chondroitin sulfate B) orheparin. After 2 hours at 37° C., the wells were washed 3× with PBS, thesupernatant removed and the number of adherent cells measured by crystalviolet staining. All values were corrected for nonspecific adhesion tomicrotiter wells coated with BSA.

FIG. 12 shows the results of a chondrocyte differentiation assay.

FIG. 13 shows the results of a collagen II staining assay.

FIG. 14 shows the results of a cartilage matrix breakdown assay. Thedata illustrated shows that WISP-3 decreases cartliage matrix breakdown.Articular cartilage explants were treated with media alone (−)′ or with150 ng/ml WISP-3 ((WISP3—), or in media with IL-1alpha at 1 ng/ml alone(+) or IL-1alpha plus WISP-3 (WISP3+) for 3 days. Cartilage matrixbreakdown was determined by measuring the amount of proteoglycans in themedia using the DMMB assay.

FIGS. 15A-15B show that WISP-1 inhibits cartilage matrix breakdown andproduction of nitric oxide. Articular cartilage explants were treatedwith media alone (−) or with 1.1 nM WISP-1 (WISP1−), or in media withIL-1alpha at 1 ng/ml alone (+) or IL-1alpha with WISP-1 (WISP1+) for 3days. In FIG. 15A, cartilage matrix breakdown was determined bymeasuring the amount of proteoglycans in the media using the DMMB assay.In FIG. 15B, nitric oxide (NO) production was determined by measuringthe amount of NO in the media using the Griess reaction.

FIG. 16 shows the skeletal phenotype of transgenic mice whichoverexpress WISP-2. Histological sections of the femur of 14 week oldwild-type (right panel) or transgenic (left panel) mice whichoverexpress WISP-2 in their skeletal muscle are shown. Note theexpansion of the zones of hyaline cartilage, namely the growth plate andthe articular cartilage, in the transgenic mice relative to those of thewild-type mice. In addition, areas of cartilage matrix appear to bepresent in the cortical bone of the transgenics, but not the wild-typemice.

FIG. 17 shows the amino acid sequences for human WISP-1-IgG (SEQ IDNO:1); mouse WISP-1-IgG (SEQ ID NO:2); “wild-type” human WISP-1 (SEQ IDNO:3); “wild-type” mouse WISP-1 (SEQ ID NO:4); and human IgG tag (SEQ IDNO:5).

FIG. 18 shows the amino acid sequences for WISP-3-IgG (SEQ ID NO:6);“alternate” WISP-3-IgG (SEQ ID NO:7); WISP-3 (SEQ ID NO:8); andWISP-3-“long 5′splicing” (SEQ ID NO:9).

FIG. 19 shows the amino acid sequence for human WISP-2 (SEQ ID NO:10).

DETAILED DESCRIPTION OF THE INVENTION

I. Definitions

The term “WISP polypeptide” refers to the family of native-sequencehuman and mouse WISP proteins and variants described herein whose genesare induced at least by Wnt-1. This term includes WISP-1, WISP-2, andWISP-3 and variants thereof. Such WISP-1, WISP-2 and WISP-3 proteins aredescribed further below and in PCT application WO99/21998 published May6, 1999 and in Pennica et al., Proc. Natl. Acad. Sci., 95:14717-14722(1998).

The terms “WISP-1 polypeptide”, “WISP-1 homologue” and grammaticalvariants thereof, as used herein, encompass native-sequence WISP-1protein and variants (which are further defined herein). The WISP-1polypeptide may be isolated from a variety of sources, such as fromhuman tissue types or from another source, or prepared by recombinant orsynthetic methods, or by any combination of these and similartechniques.

The terms “WISP-2 polypeptide”, “WISP-2 homologue”, “PRO261”, and“PRO261 polypeptide” and grammatical variants thereof, as used herein,encompass native-sequence WISP-2 protein and variants (which are furtherdefined herein). The WISP-2 polypeptide may be isolated from a varietyof sources, such as from human tissue types or from another source, orprepared by recombinant or synthetic methods, or by any combination ofthese and similar techniques.

The terms “WISP-3 polypeptide”, “WISP-3 homologue”, and grammaticalvariants thereof, as used herein, encompass native-sequence WISP-3protein and variants (which are further defined herein). The WISP-3polypeptide may be isolated from a variety of sources, such as fromhuman tissue types or from another source, or prepared by recombinant orsynthetic methods, or by any combination of these and similartechniques.

A “native-sequence WISP-1 polypeptide” comprises a polypeptide havingthe same amino acid sequence as a WISP-1 polypeptide derived fromnature. Such native-sequence WISP-1 polypeptides can be isolated fromnature or can be produced by recombinant or synthetic means. The term“native-sequence WISP-1 polypeptide” specifically encompasses naturallyoccurring truncated or secreted forms of a WISP-1 polypeptide disclosedherein, naturally occurring variant forms (e.g., alternatively splicedforms or splice variants), and naturally occurring allelic variants of aWISP-1 polypeptide. In one embodiment of the invention, thenative-sequence WISP-1 polypeptide is a mature or full-lengthnative-sequence human WISP-1 polypeptide comprising amino acids 23 to367 of SEQ ID NO:3 herein (also provided previously in FIGS. 3A and 3B(SEQ ID NO:3) shown in WO99/21998 published May 6, 1999) or amino acids1 to 367 of SEQ ID NO:3 herein (previously provided in FIGS. 3A and 3B(SEQ ID NO:4) shown in WO99/21998), respectively, with or without aN-terminal methionine. Optionally, the human WISP-1 polypeptidecomprises the contiguous sequence of amino acids 23 to 367 or aminoacids 1 to 367 of SEQ ID NO:3 herein. Optionally, the human WISP-1polypeptide is encoded by a polynucleotide sequence having the codingnucleotide sequence as in ATCC deposit no. 209533.

In another embodiment of the invention, the native-sequence WISP-1polypeptide is the full-length or mature native-sequence human WISP-1polypeptide comprising amino acids 23 to 367 or 1 to 367 of SEQ ID NO:3herein wherein the valine residue at position 184 or the alanine residueat position 202 has/have been changed to an isoleucine or serineresidue, respectively, with or without a N-terminal methionine. Inanother embodiment of the invention, the native-sequence WISP-1polypeptide is the full-length or mature native-sequence human WISP-1polypeptide comprising amino acids 23 to 367 or 1 to 367 of SEQ ID NO:3herein wherein the valine residue at position 184 and the alanineresidue at position 202 has/have been changed to an isoleucine or serineresidue, respectively, with or without a N-terminal methionine. Inanother embodiment of the invention, the native-sequence WISP-1polypeptide is a mature or full-length native-sequence mouse WISP-1polypeptide comprising amino acids 23 to 367 of SEQ ID NO:4 herein(previously provided in FIG. 1 (SEQ ID NO:11) shown in WO99/21998), oramino acids 1 to 367 of SEQ ID NO:4 herein (previously provided in FIG.1 (SEQ ID NO:12) shown in WO99/21998), respectively, with or without aN-terminal methionine.

In another embodiment of the invention, the native-sequence WISP-1polypeptide is one which is encoded by a nucleotide sequence comprisingone of the human WISP-1 splice or other native-sequence variants,including SEQ ID NOS:23, 24, 25, 26, 27, 28, or 29 shown in WO99/21998,with or without a N-terminal methionine.

A “native-sequence WISP-2 polypeptide” or a “native-sequence PRO261polypeptide” comprises a polypeptide having the same amino acid sequenceas a WISP-2 polypeptide derived from nature. Such native-sequence WISP-2polypeptides can be isolated from nature or can be produced byrecombinant or synthetic means. The term “native-sequence WISP-2polypeptide” specifically encompasses naturally occurring truncated orsecreted forms of a WISP-2 polypeptide disclosed herein, naturallyoccurring variant forms (e.g., alternatively spliced forms or splicevariants), and naturally occurring allelic variants of a WISP-2polypeptide. In one embodiment of the invention, the native-sequenceWISP-2 polypeptide is a mature or full-length native-sequence humanWISP-2 polypeptide comprising amino acids 1-24 up to 250 of SEQ ID NO:10herein (previously provided in FIG. 4 (SEQ ID NOS:15, 16, and 56-77)shown in WO99/21998), including amino acids 24 to 250 and amino acids 1to 250 of SEQ ID NO:10 herein, with or without a N-terminal methionine.Optionally, the human WISP-2 polypeptide comprises the contiguoussequence of amino acids 24 to 250 or amino acids 1 to 250 of SEQ IDNO:10 herein. Optionally, the human WISP-2 polypeptide is encoded by apolynucleotide sequence having the coding nucleotide sequence as in ATCCdeposit no. 209391. In another embodiment of the invention, thenative-sequence WISP-2 polypeptide is a mature or full-lengthnative-sequence mouse WISP-2 polypeptide comprising amino acids 1-24 upto 251 of the FIG. 2 (SEQ ID NOS:19, 20, and 78-99) shown in WO99/21998,including amino acids 24 to 251 and amino acids 1 to 251 of the FIG. 2(SEQ ID NOS:19 and 20, respectively) shown in WO99/21998, with orwithout a N-terminal methionine.

A “native-sequence WISP-3 polypeptide” comprises a polypeptide havingthe same amino acid sequence as a WISP-3 polypeptide derived fromnature. Such native-sequence WISP-3 polypeptides can be isolated fromnature or can be produced by recombinant or synthetic means. The term“native-sequence WISP-3 polypeptide” specifically encompasses naturallyoccurring truncated or other forms of a WISP-3 polypeptide disclosedherein, naturally occurring variant forms (e.g., alternatively splicedforms or splice variants), and naturally occurring allelic variants of aWISP-3 polypeptide. In one embodiment of the invention, thenative-sequence WISP-3 polypeptide is a mature or full-length,native-sequence human WISP-3 polypeptide comprising amino acids 34 to372 of SEQ ID NO:9 herein (previously provided in. 6A and 6B (SEQ IDNO:32) of WO99/21998) or amino acids 1 to 372 of SEQ ID NO:9 herein(previously provided in FIGS. 6A and 6B (SEQ ID NO:33) shown inWO99/21998), respectively, with or without a N-terminal methionine. Inanother embodiment of the invention, the native-sequence WISP-3polypeptide is a mature or full-length, native-sequence human WISP-3polypeptide comprising amino acids 16 to 354 of SEQ ID NO:8 herein(previously provided in FIGS. 7A and 7B (SEQ ID NO:36) shown in WO99/21998) or amino acids 1 to 354 of SEQ ID NO:8 herein (previouslyprovided in FIGS. 7A and 7B (SEQ ID NO:37) shown in WO99/21998),respectively, with or without a N-terminal methionine. Optionally, thehuman WISP-3 polypeptide comprises the contiguous sequence of aminoacids 34 to 372 or amino acids 1 to 372 of SEQ ID NO:9 herein.Optionally, the human WISP-3 polypeptide comprises the contiguoussequence of amino acids 16 to 354 or 1 to 354 of SEQ ID NO:8 herein.Optionally, the human WISP-3 polypeptide is encoded by a polynucleotidesequence having the coding nucleotide sequence as in ATCC deposit no.209707.

The term “WISP-1 variant” means an active WISP-1 polypeptide as definedbelow having at least about 80%, preferably at least about 85%, morepreferably at least about 90%, most preferably at least about 95% aminoacid sequence identity with human mature WISP-1 having the deduced aminoacid sequence of amino acids 23 to 367 of SEQ ID NO:3, and/or with humanfull-length WISP-1 having the deduced amino acid sequence of amino acids1 to 367 of SEQ ID NO:3, and/or with mouse mature WISP-1 having thededuced amino acid sequence shown in FIG. 1 (SEQ ID NO:11) shown inWO99/21998 and/or with mouse full-length WISP-2 having the deduced aminoacid sequence shown in the FIG. 1 (SEQ ID NO:12) of WO99/21998. Suchvariants include, for instance, WISP-1 polypeptides wherein one or moreamino acid residues are added to, or deleted from (i.e., fragments), theN- or C-terminus of the full-length or mature sequences of SEQ ID NO:3,including variants from other species, but excludes a native-sequenceWISP-1 polypeptide.

The term “WISP-2 variant” or “PRO261 variant” means an active WISP-2polypeptide as defined below having at least about 80%, preferably atleast about 85%, more preferably at least about 90%, most preferably atleast about 95% amino acid sequence identity with human mature WISP-2having the putative deduced amino acid sequence of amino acids 24 to 250of SEQ ID NO:10, and/or with human full-length WISP-2 having the deducedamino acid sequence of amino acids 1 to 250 of SEQ ID NO:10, and/or withmouse mature WISP-2 having the putative deduced amino acid sequenceshown in FIG. 2 (SEQ ID NO:19) of WO99/21998, and/or with mousefull-length WISP-2 having the deduced amino acid sequence shown in FIG.2 (SEQ ID NO:20) of WO99/21998. Such variants include, for instance,WISP-2 polypeptides wherein one or more amino acid residues are addedto, or deleted from (i.e., fragments), the N- or C-terminus of thefull-length and putative mature sequences of SEQ ID NO:10, includingvariants from other species, but excludes a native-sequence WISP-2polypeptide.

The term “WISP-3 variant” means an active WISP-3 polypeptide as definedbelow having at least about 80%, preferably at least about 85%, morepreferably at least about 90%, most preferably at least about 95% aminoacid sequence identity with human mature WISP-3 having the deduced aminoacid sequence of amino acids 34 to 372 of SEQ ID NO:9, and/or with humanfull-length WISP-3 having the deduced amino acid sequence of amino acids1 to 372 of SEQ ID NO:9, and/or with human mature WISP-3 having thededuced amino acid sequence of amino acids 16 to 354 of SEQ ID NO:8, orwith human full-length WISP-3 having the deduced amino acid sequence ofamino acids 1 to 354 of SEQ ID NO:8. Such variants include, forinstance, WISP-3 polypeptides wherein one or more amino acid residuesare added to, or deleted from (i.e., fragments), the N- or C-terminus ofthe full-length or mature sequences of SEQ ID NO:9 or SEQ ID NO:8,including variants from other species, but excludes a native-sequenceWISP-3 polypeptide.

“Percent (%) amino acid sequence identity” with respect to the WISPpolypeptide sequences identified herein is defined as the percentage ofamino acid residues in a candidate sequence that are identical with theamino acid residues in such WISP sequences identified herein, afteraligning the sequences and introducing gaps, if necessary, to achievethe maximum percent sequence identity, and not considering anyconservative substitutions as part of the sequence identity. Alignmentfor purposes of determining percent amino acid sequence identity can beachieved in various ways that are within the skill in the art, forinstance, using publicly available computer software such as BLAST,BLAST-2, ALIGN, ALIGN-2 or Megalign (DNASTAR) software. Those skilled inthe art can determine appropriate parameters for measuring alignment,including any algorithms needed to achieve maximal alignment over thefull-length of the sequences being compared. For purposes herein,however, % amino acid sequence identity values are obtained by using thesequence comparison computer program ALIGN-2. The ALIGN-2 sequencecomparison computer program was authored by Genentech, Inc. and thesource code has been filed with user documentation in the U.S. CopyrightOffice, Washington D.C., 20559, where it is registered under U.S.Copyright Registration No. TXU510087. The ALIGN-2 program is publiclyavailable through Genentech, Inc., South San Francisco, Calif. TheALIGN-2 program should be compiled for use on a UNIX operating system,preferably digital UNIX V4.0D. All sequence comparison parameters areset by the ALIGN-2 program and do not vary.

“Stringent conditions” are those that (1) employ low ionic strength andhigh temperature for washing, 0.015 M sodium chloride/0.0015 M sodiumcitrate/0.1% sodium dodecyl sulfate at 50° C.; (2) employ duringhybridization a denaturing agent, such as formamide, 50% (vol/vol)formamide with 0.1% bovine serum albumin/0.1% Ficoll/0.1%polyvinylpyrrolidone/50 mM sodium phosphate buffer at pH 6.5 with 750 mMsodium chloride, 75 mM sodium citrate at 42° C.; (3) employ 50%formamide, 5×SSC (0.75 M NaCl, 0.075 M sodium citrate), 50 mM sodiumphosphate (pH 6.8), 0.1% sodium pyrophosphate, 5× Denhardt's solution,sonicated salmon sperm DNA (50 μg/ml), 0.1% SDS, and 10% dextran sulfateat 42° C., with washes at 42° C. in 0.2×SSC and 0.1% SDS; or (4) employa buffer of 10% dextran sulfate, 2×SSC (sodium chloride/sodium citrate),and 50% formamide at 55° C., followed by a high-stringency washconsisting of 0.1×SSC containing EDTA at 55° C.

“Moderately stringent conditions” are described in Sambrook et al.,Molecular Cloning: A Laboratory Manual (New York: Cold Spring HarborLaboratory Press, 1989), and include the use of a washing solution andhybridization conditions (e.g., temperature, ionic strength, and percentSDS) less stringent than described above. An example of moderatelystringent conditions is a condition such as overnight incubation at 37°C. in a solution comprising: 20% formamide, 5×SSC (150 mM NaCl, 15 mMtrisodium citrate), 50 mM sodium phosphate (pH 7.6), 5× Denhardt'ssolution, 10% dextran sulfate, and 20 mg/mL denatured sheared salmonsperm DNA, followed by washing the filters in 1×SSC at about 37-50° C.The skilled artisan will recognize how to adjust the temperature, ionicstrength, etc., as necessary to accommodate factors such as probe lengthand the like.

“Isolated,” when used to describe the various polypeptides disclosedherein, means polypeptide that has been identified and separated and/orrecovered from a component of its natural environment. Contaminantcomponents of its natural environment are materials that would typicallyinterfere with diagnostic or therapeutic uses for the polypeptide, andmay include enzymes, hormones, and other proteinaceous ornon-proteinaceous solutes. In preferred embodiments, the polypeptidewill be purified (1) to a degree sufficient to obtain at least 15residues of N-terminal or internal amino acid sequence by use of aspinning cup sequenator, or (2) to homogeneity by SDS-PAGE undernon-reducing or reducing conditions using Coomassie blue or, preferably,silver stain. Isolated polypeptide includes polypeptide in situ withinrecombinant cells, since at least one component of the WISP naturalenvironment will not be present ordinarily, however, isolatedpolypeptide will be prepared by at least one purification step.

The term “control sequences” refers to DNA sequences necessary for theexpression of an operably linked coding sequence in a particular hostorganism. The control sequences that are suitable for prokaryotes, forexample, include a promoter, optionally an operator sequence, and aribosome binding site. Eukaryotic cells are known to utilize promoters,polyadenylation signals, and enhancers.

Nucleic acid is “operably linked” when it is placed into a functionalrelationship with another nucleic acid sequence. For example, DNA for apresequence or secretory leader is operably linked to DNA for apolypeptide if it is expressed as a preprotein that participates in thesecretion of the polypeptide; a promoter or enhancer is operably linkedto a coding sequence if it affects the transcription of the sequence; ora ribosome binding site is operably linked to a coding sequence if it ispositioned so as to facilitate translation. Generally, “operably linked”means that the DNA sequences being linked are contiguous, and, in thecase of a secretory leader, contiguous and in reading phase. However,enhancers do not have to be contiguous. Linking is accomplished byligation at convenient restriction sites. If such sites do not exist,the synthetic oligonucleotide adaptors or linkers are used in accordancewith conventional practice.

A “liposome” is a small vesicle-composed of various types of lipids,phospholipids and/or surfactant which is useful for delivery of a drug(such as the WISP polypeptides and WISP variants disclosed herein) to amammal. The components of the liposome are commonly arranged in abilayer formation, similar to the lipid arrangement of biologicalmembranes.

As used herein, the term “immunoadhesin” designates antibody-likemolecules which combine the binding specificity of a heterologousprotein (an “adhesin”) with the effector functions of immunoglobulinconstant domains. Structurally, the immunoadhesins comprise a fusion ofan amino acid sequence with the desired binding specificity which isother than the antigen recognition and binding site of an antibody(i.e., is “heterologous”), and an immunoglobulin constant domainsequence. The adhesin part of an immunoadhesin molecule typically is acontiguous amino acid sequence comprising at least the binding site of areceptor or a ligand. The immunoglobulin constant domain sequence in theimmunoadhesin may be obtained from any immunoglobulin, such as IgG-1,IgG-2, IgG-3, or IgG-4 subtypes, IgA (including IgA-1 and IgA-2), IgE,IgD or IgM.

“Active” or “activity” in the context of the WISP polypeptides or WISPvariants of the invention refers to form(s) of proteins of the inventionwhich retain the biologic and/or immunologic activities of a native ornaturally-occurring WISP polypeptide, wherein “biological” activityrefers to a biological function (either inhibitory or stimulatory)caused by a native or naturally-occurring WISP polypeptide other thanthe ability to serve as an antigen in the production of an antibodyagainst an antigenic epitope possessed by a native ornaturally-occurring polypeptide of the invention. Similarly, an“immunological” activity refers to the ability to serve as an antigen inthe production of an antibody against an antigenic epitope possessed bya native or naturally-occurring polypeptide of the invention.

“Biological activity” in the context of a WISP polypeptide or WISPvariant herein is used to refer to the ability of such molecules topromote the regeneration of and/or prevent the destruction of cartilageor to enhance or promote chondrocyte differentiation or proliferation(i.e., differentiation of a precursor cell into a mature chondrocyte).Optionally, the cartilage is articular cartilage and the regenerationand/or destruction of the cartilage is associated with an injury or adegenerative cartilagenous disorder. For example, such biologicalactivity may be quantified by the inhibition of proteoglycan (PG)release from articular cartilage, the increase in PG synthesis inarticular cartilage, the inhibition of the production of NO, etc.

The term “cartilagenous disorder” refers generally to a diseasemanifested by symptoms of pain, stiffness and/or limitation of motion ofthe affected body parts. Included within the scope of “cartilagenousdisorder” is “degenerative cartilagenous disorder”—a disordercharacterized, at least in part, by degeneration or metabolicderangement of connective tissues of the body, including not only thejoints or related structures, including muscles, bursae (synovialmembrane), tendons and fibrous tissue, but also the growth plate. In oneembodiment, the term includes “articular cartilage disorder” which ischaracterized by disruption of the smooth articular cartilage surfaceand degradation of the cartilage matrix. Additional pathologies includenitric oxide production, and inhibition or reduction of matrixsynthesis.

Included within the scope of “articular cartilage disorder” areosteoarthritis (OA) and rheumatoid arthritis (RA). OA is characterizedby localized asymmetric destruction of the cartilage commensurate withpalpable bony enlargements at the joint margins. OA typically affectsthe interphalangeal joints of the hands, the first carpometacarpaljoint, the hips, the knees, the spine, and some joints in the midfoot,while large joints, such as the ankles, elbows and shoulders tend to bespared. OA can be associated with metabolic diseases such ashemochromatosis and alkaptonuria, developmental abnormalities such asdevelopmental dysplasia of the hips (congenital dislocation of thehips), limb-length discrepancies, including trauma and inflammatoryarthritides such as gout, septic arthritis, neuropathic arthritis. OAmay also develop after extended biomechanical instability, such asresulting from sports injury or obesity.

Rheumatoid arthritis (RA) is a systemic, chronic, autoimmune disordercharacterized by symmetrical synovitis of the joint and typicallyaffects small and large diarthroid joints alike. As RA progresses,symptoms may include fever, weight loss, thinning of the skin,multiorgan involvement, scleritis, corneal ulcers, the formation ofsubcutaneous or subperiosteal nodules and even premature death. Thesymptoms of RA often appear during youth and can include vasculitis,atrophy of the skin and muscle, subcutaneous nodules, lymphadenopathy,splenomegaly, leukopaenia and chronic anaemia.

Furthermore, the term “degenerative cartilagenous disorder” may includesystemic lupus erythematosus and gout, amyloidosis or Felty's syndrome.Additionally, the term covers the cartilage degradation and destructionassociated with psoriatic arthritis, osteoarthrosis, acute inflammation(e.g., yersinia arthritis, pyrophosphate arthritis, gout arthritis(arthritis urica), septic arthritis), arthritis associated with trauma,ulcerative colitis (e.g., Crohn's disease), multiple sclerosis, diabetes(e.g., insulin-dependent and non-insulin dependent), obesity, giant cellarthritis and Sjögren's syndrome.

Examples of other immune and inflammatory diseases, at least some ofwhich may be treatable by the methods of the invention include, juvenilechronic arthritis, spondyloarthropathies, systemic sclerosis(scleroderma), idiopathic inflammatory myopathies (dermatomyositis,polymyositis), Sjögren's syndrome, systemic vasculitis, sarcoidosis,autoimmune hemolytic anemia (immune pancytopenia, paroxysmal nocturnalhemoglobinuria), autoimmune thrombocytopenia (idiopathicthrombocytopenic purpura, immune-mediated thrombocytopenia), thyroiditis(Grave's disease, Hashimoto's thyroiditis, juvenile lymphocyticthyroiditis, atrophic thyroiditis) autoimmune inflammatory diseases(e.g., allergic encephalomyelitis, multiple sclerosis, insulin-dependentdiabetes mellitus, autoimmune uveoretinitis, thyrotoxicosis,scleroderma, systemic lupus erythematosus, rheumatoid arthritis,inflammatory bowel disease (e.g., Crohn's disease, ulcerative colitis,regional enteritis, distal ileitis, granulomatous enteritis, regionalileitis, terminal ileitis), autoimmune thyroid disease, perniciousanemia) and allograft rejection, diabetes mellitus, immune-mediatedrenal disease (glomerulonephritis, tubulointerstitial nephritis),demyelinating diseases of the central and peripheral nervous systemssuch as multiple sclerosis, idiopathic demyelinating polyneuropathy orGuillain-Barre syndrome, and chronic inflammatory demyelinatingpolyneuropathy, hepatobiliary diseases such as infectious hepatitis(hepatitis A, B, C, D, E and other non-hepatotropic viruses), autoimmunechronic active hepatitis, primary biliary cirrhosis, granulomatoushepatitis, and sclerosing cholangitis, inflammatory bowel disease(ulcerative colitis, Crohn's disease), gluten-sensitive enteropathy, andWhipple's disease, autoimmune or immune-mediated skin diseases includingbullous skin diseases, erythema multiforme and contact dermatitis,psoriasis, allergic diseases such as asthma, allergic rhinitis, atopicdermatitis, food hypersensitivity and urticaria, immunologic diseases ofthe lung such as eosinophilic pneumonias, idiopathic pulmonary fibrosisand hypersensitivity pneumonitis, transplantation associated diseasesincluding graft rejection and graft-versus-host-disease. Infectiousdiseases including viral diseases such as AIDS (HIV infection),hepatitis A, B, C, D, and E, herpes, etc., bacterial infections, fungalinfections, protozoal infections, parasitic infections, and respiratorysyncytial virus, human immunodeficiency virus, etc.) and allergicdisorders, such as anaphylactic hypersensitivity, asthma, allergicrhinitis, atopic dermatitis, vernal conjunctivitis, eczema, urticariaand food allergies, etc.

“Treatment” is an intervention performed with the intention ofpreventing the development or altering the pathology of a disorder.Accordingly, “treatment” refers to both therapeutic treatment andprophylactic or preventative measures, wherein the object is to preventor slow down (lessen) the targeted pathological condition or disorder.Those in need of treatment include those already with the disorder aswell as those in which the disorder is to be prevented. In treatment ofa degenerative cartilagenous disorder, a therapeutic agent may directlydecrease or increase the magnitude of response of a pathologicalcomponent of the disorder, or render the disease more susceptible totreatment by other therapeutic agents, e.g. antibiotics, antifungals,anti-inflammatory agents, chemotherapeutics, etc.

The term “effective amount” is the minimum concentration of WISPpolypeptide which causes, induces or results in either a detectableimprovement or repair in damaged cartilage or a measureable protectionfrom the continued or induced cartilage destruction in an isolatedsample of cartilage matrix (e.g., retention of proteoglycans in matrix,inhibition of proteoglycan release from matrix, stimulation ofproteoglycan synthesis). Furthermore a “therapeutically effectiveamount” is the minimum concentration (amount) of WISP polypeptideadministered to a mammal which would be effective in at leastattenuating a pathological symptom (e.g. causing, inducing or resultingin either a detectable improvement or repair in damaged articularcartilage or causing, inducing or resulting in a measurable protectionfrom the continued or initial cartilage destruction, improvement inrange of motion, reduction in pain, etc.) which occurs as a result ofinjury or a degenerative cartilagenous disorder.

“Cartilage agent” may be a growth factor, cytokine, small molecule,antibody, piece of RNA or DNA, virus particle, peptide, or chemicalhaving a beneficial effect upon cartilage, including peptide growthfactors, catabolism antagonists and osteo-, synovial- oranti-inflammatory factors. Alternatively, “cartilage agent” may be apeptide growth factor—such as any of the fibroblast growth factors(e.g., FGF-1, FGF-2, . . . FGF-21, etc.), IGF's (I and II), TGF-βs(1-3), BMPs (1-7), or members of the epidermal growth factor family suchas EGF, HB-EGF, TGF-β—which could enhance the intrinsic reparativeresponse of cartilage, for example by altering proliferation,differentiation, migration, adhesion, or matrix production bychondrocytes. Alternatively, a “cartilage agent” may be a factor whichantagonizes the catabolism of cartilage (e.g., IL-1 receptor antagonist(IL-1ra), NO inhibitors, IL1-beta convertase (ICE) inhibitors, factorswhich inhibit activity of IL-6, IL-8, LIF, IFN-gamma, or TNF-alphaactivity, tetracyclines and variants thereof, inhibitors of apoptosis,MMP inhibitors, aggrecanase inhibitors, inhibitors of serine andcysteine proteinases such as cathepsins and urokinase or tissueplasminogen activator (uPA and tPA). Alternatively still, cartilageagent includes factors which act indirectly on cartilage by affectingthe underlying bone (i.e., osteofactors, e.g. bisphosphonates orosteoprotegerin) or the surrounding synovium (i.e., synovial factors) oranti-inflammatory factors (e.g., anti-TNF-alpha (includinganti-TNF-alpha antibodies such as Remicade®, as well as TNF receptorimmunoadhesins such as Enbrel®), IL-1ra, IL-4, IL-10, IL-13, NSAIDs).For a review of cartilage agent examples, please see Martel-Pelletier etal., Front. Biosci. 4: d694-703 (1999); Hering, T. M., Front. Biosci. 4:d743-761 (1999).

“Chronic” administration refers to administration of the factor(s) in acontinuous mode as opposed to an acute mode, so as to maintain theinitial therapeutic effect (activity) for an extended period of time.

“Intermittent” administration is treatment that is done notconsecutively without interruption, but rather is cyclic in nature.

“Mammal” for purposes of treatment refers to any animal classified as amammal, including humans, domestic and farm animals, and zoo, sports, orpet animals, such as dogs, horses, cats, cattle, pigs, hamsters, etc.Preferably, the mammal is human.

Administration “in combination with” one or more further therapeuticagents includes simultaneous (concurrent) and consecutive administrationin any order.

“Carriers” as used herein include pharmaceutically acceptable carriers,excipients, or stabilizers which are nontoxic to the cell or mammalbeing exposed thereto at the dosages and concentrations employed. Oftenthe physiologically acceptable carrier is an aqueous pH bufferedsolution. Examples of physiologically acceptable carriers includebuffers such as phosphate, citrate, and other organic acids;antioxidants including ascorbic acid; low molecular weight (less thanabout 10 residues) polypeptide; proteins, such as serum albumin,gelatin, or immunoglobulins; hydrophilic polymers such aspolyvinylpyrrolidone; amino acids such as glycine, glutamine,asparagine, arginine or lysine; monosaccharides, disaccharides, andother carbohydrates including glucose, mannose, or dextrins; chelatingagents such as EDTA; sugar alcohols such as mannitol or sorbitol;salt-forming counterions such as sodium; and/or nonionic surfactantssuch as TWEEN®, polyethylene glycol (PEG), and PLURONICS®, hyaluronicacid (HA).

II. Methods and Compositions of the Invention

In accordance with the methods of the present invention, various WISPpolypeptides may be employed for treatment of degenerative cartilagenousdisorders as well as various other immune and immune related conditions.Such WISP polypeptides include the polypeptides referred to herein asWISP-1, WISP-2, and WISP-3 and variants thereof (as well as fusionproteins thereof such as epitope tagged forms or Ig-fusion constructsthereof). The WISP polypeptides may be used in vivo as well as ex vivo.Optionally, the WISP polypeptides are used in the form of pharmaceuticalcompositions, described in further detail below.

Degenerative cartilagenous disorders contemplated by the inventioninclude Rheumatoid arthritis (RA). RA is a systemic, autoimmune,degenerative disease that can cause symmetrical disruptions in thesynovium of both large and small diarthroidal joints. As the diseaseprogresses, symptoms of PA may include fever, weight loss, thinning ofthe skin, multiorgan involvement, scleritis, corneal ulcers, formationof subcutaneous or subperiosteal nodules and premature death. RAsymptoms typically appear during youth, extra-articular manifestationscan affect any organ system, and joint destruction is symmetrical andoccurs in both large and small joints alike. Extra-articular symptomscan include vasculitis, atrophy of the skin and muscle, subcutaneousnodules, lymphadenopathy, splenomegaly, leukopaenia and chronic anaemia.RA tends to be heterogeneous in nature with a variable diseaseexpression and is associated with the formation of serum rheumatoidfactor in 90% of patients sometime during the course of the illness. RApatients typically also have a hyperactive immune system. The majorityof people with RA have a genetic susceptibility associated withincreased activation of class II major histocompatibility complexmolecules on monocytes and macrophages. These histocompatibility complexmolecules are involved in the presentation of antigen to activated Tcells bearing receptors for these class II molecules. The geneticpredisposition to RA is supported by the prevalence of the highlyconserved leukocyte antigen DR subtype Dw4, Dw14 and Dw15 in humanpatients with very severe disease.

Osteoarthritis (OA) is another degenerative cartilagenous disorder thatinvolves a localized disease that affects articular cartilage and boneand results in pain and diminished joint function. OA may be classifiedinto two types: primary and secondary. Primary OA refers to the spectrumof degenerative joint diseases for which no underlying etiology has beendetermined. Typically, the joint affected by primary OA are theinterphalangeal joints of the hands, the first carpometacarpal joints,the hips, the knees, the spine, and some joints in the midfoot. Largejoints, such as the ankles, elbows and shoulders tend to be spared inprimary OA. In contrast, secondary OA often occurs as a result ofdefined injury or trauma. Secondary arthritis can also be found inindividuals with metabolic diseases such as hemochromatosis andalkaptonuria, developmental abnormalities such as developmentaldysplasia of the hips (congenital dislocation of the hips) andlimb-length discrepancies, obesity, inflammatory arthritides such asrheumatoid arthritis or gout, septic arthritis, and neuropathicarthritis.

The degradation associated with OA initially appears as fraying andfibrillation of the articular cartilage surface as proteoglycans arelost from the matrix. With continued joint use, surface fibrillationprogresses, defects penetrate deeper into the cartilage, and pieces ofcartilage tissue are lost. In addition, bone underlying the cartilage(subchondral bone) thickens, and, as cartilage is lost, bone becomesslowly exposed. With asymmetric cartilage destruction, disfigurement canoccur. Bony nodules, called osteophytes, often form at the periphery ofthe cartilage surface and occasionally grow over the adjacent erodedareas. If the surface of these bony outgrowths is permeated, vascularoutgrowth may occur and cause the formation of tissue plugs containingfibrocartilage.

Since cartilage is avascular, damage which occurs to the cartilage layerbut does not penetrate to the subchondral bone, leaves the job of repairto the resident chondrocytes, which have little intrinsic potential forreplication. However, when the subchondral bone is penetrated, itsvascular supply allows a triphasic repair process to take place. Thesuboptimal cartilage which is synthesized in response to this type ofdamage, termed herein “fibrocartilage” because of its fibrous matrix,has suboptimal biochemical and mechanical properties, and is thussubject to further wear and destruction. In a diseased or damaged joint,increased release of metalloproteinases (MMPs) such as collagenases,gelatinases, stromelysins, aggrecanases, and other proteases, leads tofurther thinning and loss of cartilage. In vitro studies have shown thatcytokines such as IL-1alpha, IL-1beta, TNF-alpha, PDGF, GM-CSF,IFN-gamma, TGF-beta, LIF, IL-2 and IL-6, IL-8 can alter the activity ofsynovial fibroblast-like cells, macrophage, T cells, and/or osteoclasts,suggesting that these cytokines may regulate cartilage matrix turnoverin vivo.

The mechanical properties of cartilage are determined by its biochemicalcomposition. While the collagen architecture contributes to the tensilestrength and stiffness of cartilage, the compressibility (or elasticity)is due to its proteoglycan component. In healthy articular cartilage,type II collagen predominates (comprising about 90-95%), however,smaller amounts of types V, VI, IX, and XI collagen are also present.Cartilage proteoglycans (PG) include hydrodynamically large, aggregatingPG, with covalently linked sulfated glycosaminoglycans, as well ashydrodynamically smaller nonaggregating PG such as decorin, biglycan andlumican.

Injuries to cartilage may fall into three categories: (1) microdamage orblunt trauma, (2) chondral fractures, and (3) osteochondral fractures.

Microdamage to chondrocytes and cartilage matrix may be caused by asingle impact, through repetitive blunt trauma, or with continuous useof a biomechanically unstable joint. Metabolic and biochemical changessuch as those found in the early stages of degenerative arthritis can bereplicated in animal models involving repetitive loading of articularcartilage. Radin et al., Clin. Orthop. Relat. Res. 131: 288-93 (1978).Such experiments, along with the distinct pattern of cartilage lossfound in arthritic joints, highlight the role that biomechanical loadingplays in the loss of homeostasis and integrity of articular cartilage indisease. Radin et al., J Orthop Res. 2: 221-234 (1984); Radin et al.,Semin Arthritis Rheum (suppl. 2) 21: 12-21 (1991); Wei et al., ActaOrthop Scand 69: 351-357 (1998). While chondrocytes may initially beable to replenish cartilage matrix with proteoglycans at a basal rate,concurrent damage to the collagen network may increase the rate of lossand result in irreversible degeneration. Buckwalter et al., J. Am. Acad.Orthop. Surg. 2: 192-201 (1994).

Chondral fractures are characterized by disruption of the articularsurface without violation of the subchondral plate. Chondrocyte necrosisat the injury site occurs, followed by increased mitotic and metabolicactivity of the surviving chondrocytes bordering the injury which leadsto lining of the clefts of the articular surface with fibrous tissue.The increase in chondrocyte activity is transitory, and the repairresponse results in insufficient amount and quality of new matrixcomponents.

Osteochondral fractures, the most serious of the three types ofinjuries, are lesions crossing the tidemark into the underlyingsubchondral plate. In this type of injury, the presence of subchondralvasculature elicits the three-phase response typically encountered invascular tissues: (1) necrosis, (2) inflammation, and (3) repair.Initially the lesion fills with blood and clots. The resulting fibrinclot activates an inflammatory response and becomes vascularized repairtissue, and the various cellular components release growth factors andcytokines including transforming growth factor beta (TGF-beta),platelet-derived growth factor (PDGF), bone morphogenic proteins, andinsulin-like growth factors I and II. Buckwalter et al., J. Am. Acad.Orthop. Surg. 2: 191-201 (1994).

The initial repair response associated with osteochondral fractures ischaracterized by recruitment, proliferation and differentiation ofprecursors into chondrocytes. Mesenchymal stem cells are deposited inthe fibrin network, which eventually becomes a fibrocartilagenous zone.F. Shapiro et al., J. Bone Joint Surg. 75: 532-53 (1993); N. Mitchelland N. Shepard, J. Bone Joint Surg. 58: 230-33 (1976). These stem cells,which are believed to come from the underlying bone marrow rather thanthe adjacent articular surface, progressively differentiate intochondrocytes. At six to eight weeks after injury, the repair tissuecontains chondrocyte-like cells in a matrix of proteoglycans andpredominantly type II collagen, with some type I collagen. T. Furukawaet al., J. Bone Joint Surg. 62: 79-89 (1980); J. Cheung et al.,Arthritis Rheum. 23: 211-19 (1980); S. O. Hjertquist & R. Lemperg, Calc.Tissue Res. 8: 54-72 (1971). However, this newly deposited matrixdegenerates, and the chondroid tissue is replaced by more fibrous tissueand fibrocartilage and a shift in the synthesis of collagen from type IIto type I. H. S. Cheung et al., J. Bone Joint Surg. 60: 1076-81 (1978);D. Hamerman, “Prospects for medical intervention in cartilage repair,”Joint cartilage degradation: Basic and clinical aspects, Eds. Woessner JF et al., (1993); Shapiro et al., J. Bone Joint Surg. 75: 532-53 (1993);N. Mitchell & N. Shepard, J. Bone Joint Surg. 58: 230-33 (1976); S. O.Hjertquist & R. Lemperg, Calc. Tissue Res. 8: 54-72 (1971). Earlydegenerative changes include surface fibrillation, depletion ofproteoglycans, chondrocyte cloning and death, and vertical fissuringfrom the superficial to deep layers. At one year post-injury, the repairtissue is a mixture of fibrocartilage and hyaline cartilage, with asubstantial amount of type I collagen, which is not found in appreciableamounts in normal articular cartilage. T. Furukawa, et al., J. BoneJoint Surg. 62: 79-89 (1980).

While inflammation does not appear to be the initiating event inosteoarthritis, inflammation does occur in osteoarthritic joints. Theinflammatory cells (i.e. monocytes, macrophages, and neutrophils) whichinvade the synovial lining after injury and during inflammation producemetalloproteinases as well as catabolic cyokines which can contribute tofurther release of degradative enzymes. Although inflammation and jointdestruction do not show perfect correlation in all animal models ofarthritis, agents such as IL-4, IL-10 and IL-13 which inhibitinflammation also decrease cartilage and bone pathology in arthriticanimals (reviewed in Martel-Pelletier J. et al. Front. Biosci. 4:d694-703). Application of agents which inhibit inflammatory cytokinesmay slow OA progression by countering the local synovitis which occursin OA patients.

OA involves not only the degeneration of articular cartilage leading toeburnation of bone, but also extensive remodelling of subchondral boneresulting in the so-called sclerosis of this tissue. These bony changesare often accompanied by the formation of subchondral cysts as a resultof focal resorption. Agents which inhibit bone resorption, i.e.osteoprotegerin or bisphosphonates have shown promising results inanimal model of arthritis. Kong et al. Nature 402: 304-308 (1999).

In systemic lupus erythematosus, the central mediator of disease is theproduction of auto-reactive antibodies to self proteins/tissues and thesubsequent generation of immune-mediated inflammation. These antibodieseither directly or indirectly mediate tissue injury. Although Tlymphocytes have not been shown to be directly involved in tissuedamage, T lymphocytes are required for the development of auto-reactiveantibodies. The genesis of the disease is thus T lymphocyte dependent.Multiple organs and systems are affected clinically including kidney,lung, musculoskeletal system, mucocutaneous, eye, central nervoussystem, cardiovascular system, gastrointestinal tract, bone marrow andblood.

Juvenile chronic arthritis is a chronic idiopathic inflammatory diseasewhich begins often at less than 16 years of age and which has somesimilarities to RA. Some patients which are rheumatoid factor positiveare classified as juvenile rheumatoid arthritis. The disease issub-classified into three major categories: pauciarticular,polyarticular, and systemic. The arthritis can be severe and leads tojoint ankylosis and retarded growth. Other manifestations can includechronic anterior uveitis and systemic amyloidosis.

Spondyloarthropathies are a group of disorders with some common clinicalfeatures and the common association with the expression of HLA-B27 geneproduct. The disorders include: ankylosing sponylitis, Reiter's syndrome(reactive arthritis), arthritis associated with inflammatory boweldisease, spondylitis associated with psoriasis, juvenile onsetspondyloarthropathy and undifferentiated spondyloarthropathy.Distinguishing features include sacroileitis with or withoutspondylitis; inflammatory asymmetric arthritis; association with HLA-B27(a serologically defined allele of the HLA-B locus of class I MHC);ocular inflammation, and absence of autoantibodies associated with otherrheumatoid disease. The cell most implicated as key to induction of thedisease is the CD8+ T lymphocyte, a cell which targets antigen presentedby class I MHC molecules. CD8+ T cells may react against the class I MHCallele HLA-B27 as if it were a foreign peptide expressed by MHC class Imolecules. It has been hypothesized that an epitope of HLA-B27 may mimica bacterial or other microbial antigenic epitope and thus induce a CD8+T cells response.

The WISP polypeptides employed in the invention may be prepared by anysuitable method, including recombinant expresssion techniques.Recombinant expression technology is well known to those skilled in theart, and optional materials and methods are described in PCTapplication, WO 99/21998. Optionally, the WISP polypeptides areexpressed using host cell such as CHO cells, E. coli or yeast cells. TheWISP polypeptides may comprise full length polypeptides (definedherein), or variant forms thereof, as well as other modified forms ofthe WISP polypeptides (such as by fusing or linking to animmunoglobulin, epitope tag, leucine zipper or other non-proteinaceouspolymer).

Immunoadhesin molecules are contemplated for use in the methods herein.WISP immunoadhesins may comprise various forms of WISP, such as the fulllength polypeptide as well as variant or fragment forms thereof. In oneembodiment, the molecule may comprise a fusion of the WISP with animmunoglobulin or a particular region of an immunoglobulin. For abivalent form of the immunoadhesin, such a fusion could be to the Fcregion of an IgG molecule. For the production of immunoglobulin fusions,see also U.S. Pat. No. 5,428,130 issued Jun. 27, 1995 and Chamow et al.,TIBTECH, 14:52-60 (1996).

In another embodiment, the WISP polypeptide may be covalently modifiedby linking the polypeptide to one of a variety of nonproteinaceouspolymers, e.g., polyethylene glycol (PEG), polypropylene glycol, orpolyoxyalkylenes, in the manner set forth in U.S. Pat. Nos. 4,640,835;4,496,689; 4,301,144; 4,670,417; 4,791,192 or 4,179,337. Such pegylatedforms of the WISP polypeptide may be prepared using techniques known inthe art.

Leucine zipper forms of these molecules are also contemplated by theinvention. “Leucine zipper” is a term in the art used to refer to aleucine rich sequence that enhances, promotes, or drives dimerization ortrimerization of its fusion partner (e.g., the sequence or molecule towhich the leucine zipper is fused or linked to). Various leucine zipperpolypeptides have been described in the art. See, e.g., Landschulz etal., Science, 240:1759 (1988); U.S. Pat. No. 5,716,805; WO 94/10308;Hoppe et al., FEBS Letters, 344:1991 (1994); Maniatis et al., Nature,341:24 (1989). Those skilled in the art will appreciate that a leucinezipper sequence may be fused at either the 5′ or 3′ end of the WISPpolypeptide.

The WISP polypeptides of the present invention may also be modified in away to form chimeric molecules by fusing the receptor polypeptide toanother, heterologous polypeptide or amino acid sequence. Preferably,such heterologous polypeptide or amino acid sequence is one which actsto oligimerize the chimeric molecule. In one embodiment, such a chimericmolecule comprises a fusion of the WISP polypeptide with a tagpolypeptide which provides an epitope to which an anti-tag antibody canselectively bind. The epitope tag is generally placed at the amino- orcarboxyl-terminus of the polypeptide. The presence of suchepitope-tagged forms of the WISP polypeptide can be detected using anantibody against the tag polypeptide. Also, provision of the epitope tagenables the WISP polypeptide to be readily purified by affinitypurification using an anti-tag antibody or another type of affinitymatrix that binds to the epitope tag. Various tag polypeptides and theirrespective antibodies are well known in the art. Examples includepoly-histidine (poly-his) or poly-histidine-glycine (poly-his-gly) tags;the flu HA tag polypeptide and its antibody 12CA5 [Field et al., Mol.Cell. Biol., 8:2159-2165 (1988)]; the c-myc tag and the 8F9, 3C7, 6E10,G4, B7 and 9E10 antibodies thereto [Evan et al., Molecular and CellularBiology, 5:3610-3616 (1985)]; and the Herpes Simplex virus glycoproteinD (gD) tag and its antibody [Paborsky et al., Protein Engineering,3(6):547-553 (1990)). Other tag polypeptides include the Flag-peptide[Hopp et al., BioTechnology, 6:1204-1210 (1988)]; the KT3 epitopepeptide [Martin et al., Science, 255:192-194 (1992)]; an α-tubulinepitope peptide [Skinner et al., J. Biol. Chem., 266:15163-15166(1991)]; and the T7 gene 10 protein peptide tag (Lutz-Freyermuth et al.,Proc. Natl. Acad. Sci. USA, 87:6393-6397 (1990)].

Formulations of WISP polypeptides employable with the invention can beprepared by mixing the WISP polypeptide having the desired degree ofpurity with optional pharmaceutically acceptable carriers, excipients orstabilizers (Remington's Pharmaceutical Sciences 16th edition, Osol, A.Ed. [1980]). Such therapeutic formulations can be in the form oflyophilized formulations or aqueous solutions. Acceptable carriers,excipients, or stabilizers are nontoxic to recipients at the dosages andconcentrations employed, and include buffers such as phosphate, citrate,and other organic acids; antioxidants including ascorbic acid andmethionine; preservatives (such as octadecyldimethylbenzyl ammoniumchloride; hexamethonium chloride; benzalkonium chloride, benzethoniumchloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methylor propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; andm-cresol); low molecular weight (less than about 10 residues)polypeptides; proteins, such as serum albumin, gelatin, orimmunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone;amino acids such as glycine, glutamine, asparagine, histidine, arginine,or lysine; monosaccharides, disaccharides, and other carbohydratesincluding glucose, mannose, dextrins, or hyaluronan; chelating agentssuch as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol;salt-forming counter-ions such as sodium; metal complexes (e.g.Zn-protein complexes); and/or non-ionic surfactants such as TWEEN®,PLURONICS® or polyethylene glycol (PEG).

The WISP polypeptides also may be prepared by entrapping inmicrocapsules prepared, for example by coacervation techniques or byinterfacial polymerization, for example, hydroxymethylcellulose orgelatin-microcapsules and poly-(methylmethacrylate) microcapsules,respectively. Such preparations can be administered in colloidal drugdelivery systems (for example, liposomes, albumin microspheres,microemulsions, nanoparticles and nanocapsules) or in macroemulsions.Such techniques are disclosed in Remington's Pharmaceutical Sciences,16th Edition (or newer), Osol A. Ed. (1980).

Where sustained-release or extended-release administration of the WISPpolypeptides is desired in a formulation with release characteristicssuitable for the treatment of any disease or disorder requiringadministration of such polypeptides, microencapsulation is contemplated.Microencapsulation of recombinant proteins for sustained release hasbeen successfully performed. See, e.g., Johnson et al., Nat. Med. 2:795-799 (1996); Yasuda, Biomed. Ther. 27: 1221-1223 (1993); Hora et al.,Bio/Technology 8: 755-758 (1990); Cleland, “Design and Production ofSingle Immunization Vaccines Using Polylactide Polyglycolide MicrosphereSystems” in Vaccine Design: The Subunit and Adjuvant Approach, Powelland Newman, eds., (Plenum Press: New York, 1995), pp. 439-462; WO97/03692, WO 96/40072, WO 96/07399 and U.S. Pat. No. 5,654,010.

Suitable examples of sustained-release preparations includesemipermeable matrices of solid hydrophobic polymers containing theactive molecule, which matrices are in the form of shaped articles, e.g.films, or microcapsules. Examples of sustained-release matrices includeone or more polyanhydrides (e.g., U.S. Pat. Nos. 4,891,225; 4,767,628),polyesters such as polyglycolides, polylactides andpolylactide-co-glycolides (e.g., U.S. Pat. No. 3,773,919; U.S. Pat. No.4,767,628; U.S. Pat. No. 4,530,840; Kulkarni et al., Arch. Surg. 93: 839(1966)), polyamino acids such as polylysine, polymers and copolymers ofpolyethylene oxide, polyethylene oxide acrylates, polyacrylates,ethylene-vinyl acetates, polyamides, polyurethanes, polyorthoesters,polyacetylnitriles, polyphosphazenes, and polyester hydrogels (forexample, poly(2-hydroxyethyl-methacrylate), or poly(vinylalcohol)),cellulose, acyl substituted cellulose acetates, non-degradablepolyurethanes, polystyrenes, polyvinyl chloride, polyvinyl fluoride,poly(vinylimidazole), chlorosulphonated polyolefins, polyethylene oxide,copolymers of L-glutamic acid and gamma-ethyl-L-glutamate,non-degradable ethylene-vinyl acetate, degradable lactic acid-glycolicacid copolymers such as the LUPRON DEPOT® (injectable microspherescomposed of lactic acid-glycolic acid copolymer and leuprolide acetate),and poly-D-(−)-3-hydroxybutyric acid. While polymers such asethylene-vinyl acetate and lactic acid-glycolic acid enable release ofmolecules for over 100 days, certain hydrogels release proteins forshorter time periods. Additional non-biodegradable polymers which may beemployed are polyethylene, polyvinyl pyrrolidone, ethylene vinylacetate,polyethylene glycol, cellulose acetate butyrate and cellulose acetatepropionate.

Alternatively, sustained release formulations may be composed ofdegradable biological materials. Biodegradable polymers are attractivedrug formulations because of their biocompatibility, high responsibilityfor specific degradation, and ease of incorporating the active drug intothe biological matrix. For example, hyaluronic acid (HA) may becrosslinked and used as a swellable polymeric delivery vehicle forbiological materials. U.S. Pat. No. 4,957,744; Valle et al., Polym.Mater. Sci. Eng. 62: 731-735 (1991). HA polymer grafted withpolyethylene glycol has also been prepared as an improved deliverymatrix which reduced both undesired drug leakage and the denaturingassociated with long term storage at physiological conditions. Kazuteru,M., J. Controlled Release 59:77-86 (1999). Additional biodegradablepolymers which may be used are poly(caprolactone), polyanhydrides,polyamino acids, polyorthoesters, polycyanoacrylates,poly(phosphazines), poly(phosphodiesters), polyesteramides,polydioxanones, polyacetals, polyketals, polycarbonates,polyorthocarbonates, degradable and nontoxic polyurethanes,polyhydroxylbutyrates, polyhydroxyvalerates, polyalkylene oxalates,polyalkylene succinates, poly(malic acid), chitin and chitosan.

Alternatively, biodegradable hydrogels may be used as controlled releasedelivery vehicles for biological materials and drugs. Through theappropriate choice of macromers, membranes can be produced with a rangeof permeability, pore sizes and degradation rates suitable for a widevariety of biomolecules.

Alternatively, sustained-release delivery systems for biologicalmaterials and drugs can be composed of dispersions. Dispersions mayfurther be classified as either suspensions or emulsions. In the contextof delivery vehicles for biological materials, suspensions are a mixtureof very small solid particles which are dispersed (more or lessuniformly) in a liquid medium. The solid particles of a suspension canrange in size from a few nanometers to hundreds of microns, and includemicrospheres, microcapsules and nanospheres. Emulsions, on the otherhand, are a mixture of two or more immiscible liquids held in suspensionby small quantities of emulsifiers. Emulsifiers form an interfacial filmbetween the immiscible liquids and are also known as surfactants ordetergents. Emulsion formulations can be both oil in water (o/w) whereinwater is in a continuous phase while the oil or fat is dispersed, aswell as water in oil (w/o), wherein the oil is in a continuous phasewhile the water is dispersed. One example of a suitablesustained-release formulation is disclosed in WO 97/25563. Additionally,emulsions for use with biological materials include multiple emulsions,microemulsions, microdroplets and liposomes. Microdroplets areunilamellar phospholipid vesicles that consist of a spherical lipidlayer with an oil phase inside. E.g., U.S. Pat. No. 4,622,219 and U.S.Pat. No. 4,725,442. Liposomes are phospholipid vesicles prepared bymixing water-insoluble polar lipids with an aqueous solution.

Alternatively, the sustained-release formulations of WISP polypeptidesmay be developed using poly-lactic-coglycolic acid (PLGA), a polymerexhibiting a strong degree of biocompatibility and a wide range ofbiodegradable properties. The degradation products of PLGA, lactic andglycolic acids, are cleared quickly from the human body. Moreover, thedegradability of this polymer can be adjusted from months to yearsdepending on its molecular weight and composition. For furtherinformation see Lewis, “Controlled Release of Bioactive Agents fromLactide/Glycolide polymer,” in Biogradable Polymers as Drug DeliverySystems M. Chasin and R. Langeer, editors (Marcel Dekker: New York,1990), pp. 1-41.

The encapsulated polypeptides or polypeptides in extended-releaseformulation may be imparted by formulating the polypeptide with a“water-soluble polyvalent metal salts” which are non-toxic at therelease concentration and temperature. Exemplary “polyvalent metals”include the following cations: Ca²⁺, Mg²⁺, Zn²⁺, Fe²⁺, Fe³⁺, Cu²⁺, Sn²⁺,Sn²⁺, Al²+ and Al³⁺. Exemplary anions which form water-soluble saltswith the above polyvalent metal cations include those formed byinorganic acids and/or organic acids. Such water-soluble salts havesolubility in water (at 20° C.) of at least about 20 mg/ml,alternatively 100 mg/ml, alternatively 200 mg/ml.

Suitable inorganic acids that can be used to form the “water solublepolyvalent metal salts” include hydrochloric, sulfuric, nitric,thiocyanic and phosphoric acid. Suitable organic acids that can be usedinclude aliphatic carboxylic acid and aromatic acids. Aliphatic acidswithin this definition may be defined as saturated or unsaturated C₂₋₉carboxylic acids (e.g., aliphatic mono-, di- and tri-carboxylic acids).Commonly employed water soluble polyvalent metal salts which may be usedto help stabilize the encapsulated polypeptides of this inventioninclude, for example: (1) the inorganic acid metal salts of halides(e.g., zinc chloride, calcium chloride), sulfates, nitrates, phosphatesand thiocyanates; (2) the aliphatic carboxylic acid metal salts calciumacetate, zinc acetate, calcium proprionate, zinc glycolate, calciumlactate, zinc lactate and zinc tartrate; and (3) the aromatic carboxylicacid metal salts of benzoates (e.g., zinc benzoate) and salicylates.

In order for the formulations to be used for in vivo administration,they should be sterile. The formulation may be readily rendered sterileby filtration through sterile filtration membranes, prior to orfollowing lyophilization and reconstitution. The therapeuticcompositions herein generally are placed into a container having asterile access port, for example, an intravenous solution bag or vialhaving a stopper pierceable by a hypodermic injection needle.

For treatment of the mammal in vivo, the route of administration is inaccordance with known methods, e.g., injection or infusion byintravenous, intraperitoneal, intramuscular, intraarterial,intralesional or intraarticular routes, topical administration, bysustained release or extended-release means. Optionally the activecompound or formulation is injected directly or locally into theafflicted cartilagenous region or articular joint. The treatmentcontemplated by the invention may also take the form of gene therapy.

Dosages' and desired drug concentrations of pharmaceutical compositionsemployable with the present invention may vary depending on theparticular use envisioned. The determination of the appropriate dosageor route of administration is well within the skill of an ordinaryphysician. Animal experiments can provide reliable guidance for thedetermination of effective doses for human therapy. Interspecies scalingof effective doses can be performed following the principles laid downby Mordenti, J. and Chappell, W. “The use of interspecies scaling intoxicokinetics” in Toxicokinetics and New Drug Development, Yacobi etal., Eds., Pergamon Press, New York 1989, pp. 42-96.

When in vivo administration of WISP polypeptides are employed, normaldosage amounts may vary from about 10 ng/kg to up to 100 mg/kg of mammalbody weight or more per day, preferably about 1 μg/kg/day to 10mg/kg/day, depending upon the route of administration. Guidance as toparticular dosages and methods of delivery is provided in theliterature; see, for example, U.S. Pat. Nos. 4,657,760; 5,206,344 or5,225,212. It is anticipated that different formulations will beeffective for different treatments and different disorders, and thatadministration intended to treat a specific organ or tissue, maynecessitate delivery in a manner different from that to another organ ortissue.

The formulations used herein may also contain more than one activecompound as necessary for the particular indication being treated,preferably those with complementary activities that do not adverselyaffect each other. The WISP polypeptide may be administered incombination with a cytotoxic agent, cytokine or growth inhibitory agent.Such molecules are present in combinations and amounts that areeffective for the intended purpose. It may be desirable to alsoadminister antibodies against other immune disease associated or tumorassociated antigens, such as antibodies which bind to CD20, CD11a, CD40, CD18, ErbB2, EGFR, ErbB3, ErbB4, or vascular endothelial growthfactor (VEGF). Alternatively, or in addition, two or more antibodiesbinding the same or two or more different antigens disclosed herein maybe coadministered to the patient. Sometimes, it may be beneficial toalso administer one or more cytokines to the patient. In one embodiment,the polypeptides of the invention are coadministered with a growthinhibitory agent. For example, the growth inhibitory agent may beadministered first, followed by a WISP polypeptide of the invention.Still other agents may be administered in combination with WISPpolypeptide, such as agents like decorin or biglycan. Simultaneousadministration or sequential administration is also contemplated.

The present method may also be administered in combination with anystandard cartilage surgical technique. Standard surgical techniques aresurgical procedures which are commonly employed for therapeuticmanipulations of cartilage, including: cartilage shaving, abrasionchondroplasty, laser repair, debridement, chondroplasty, microfracturewith or without subchondral bone penetration, mosaicplasty, cartilagecell allografts, stem cell autografts, costal cartilage grafts, chemicalstimulation, electrical stimulation, perichondral autografts, periostealautografts, cartilage scaffolds, shell (osteoarticular) autografts orallografts, or osteotomy. These techniques are described and discussedin greater detail in Frenkel et al., Front. Bioscience 4: d671-685(1999).

In a preferred embodiment, the WISP polypeptides are used in combinationwith microfracture surgery. Microfracture surgery techniques are knownin the art and generally entail surgical drilling into the mammal's bonemarrow cavity. Fibrin clots then form, filling the defect in themammals's body. Subsequently, fibrocartilage forms.

It is contemplated that WISP polypeptides can be employed to treatcartilage or chondrocyte cells ex vivo. Such ex vivo treatment may beuseful in transplantation and particularly, autologous transplantation.For instance, treatment of cells or tissue(s) containing such cartilageor chondrocyte cells with WISP polypeptide, and optionally, with one ormore other therapies, such as described above, can be employed toregenerate cartilage tissue of induce differentiation of precursorchondrocyte cells prior to transplantation in a recipient mammal.

Cells or tissue(s) containing cartilage or chondrocyte cells are firstobtained from a donor mammal. The cells or tissue(s) may be obtainedsurgically and preferably, are obtained aseptically. The cells ortissue(s) are then treated with WISP polypeptide, and optionally, withone or more other therapies, such as described above.

The treated cells or tissue(s) can then be infused or transplanted intoa recipient mammal. The recipient mammal may be the same individual asthe donor mammal or may be another, heterologous mammal.

The progress or effectiveness of the therapies described herein can bereadily monitored by conventional techniques and assays known to theskilled practicioner.

The activity or effects of the WISP polypeptides described herein oncartilage or chondrocytes can be determined without undueexperimentation using various in vitro or in vivo assays. By way ofexample, several such assays are described below.

In one assay, the synthetic and prophylactic potential of WISPpolypeptide on intact cartilage can be tested. To this end, proteoglycan(PG) synthesis and breakdown, and nitric oxide release are measured intreated articular cartilage explants. Proteoglycans are the secondlargest component of the organic material in articular cartilage(Kuettner, K. E. et al., Articular Cartilage Biochemistry, Raven Press,New York, USA (1986), p.456; Muir, H., Biochem. Soc. Tran. 11: 613-622(1983); Hardingham, T. E., Biochem. Soc. Trans. 9: 489-497 (1981). Sinceproteoglycans help determine the physical and chemical properties ofcartilage, the decrease in cartilage PGs which occurs during jointdegeneration leads to loss of compressive stiffness and elasticity, anincrease in hydraulic permeability, increased water content (swelling),and changes in the organization of other extracellular components suchas collagens. Thus, PG loss is an early step in the progression ofdegenerative cartilaginous disorders, one which further perturbs thebiomechanical and biochemical stability of the joint. PGs in articularcartilage have been extensively studied because of their likely role inskeletal growth and disease. Mow, V. C., & Ratcliffe, A. Biomaterials13: 67-97 (1992). Proteoglycan breakdown, which is increased in diseasedjoints, can be measured by quantitating PGs released into the media byarticular cartilage explants using the colorimetric DMMB assay. Farndaleand Buttle, Biochem. Biophys. Acta 883: 173-177 (1985). Incorporation of³⁵S-sulfate into proteoglycans is used to measure proteoglycansynthesis.

The evidence linking interleukin-1alpha, IL-1beta, and degenerativecartilagenous diseases is substantial. For example, high levels ofIL-1alpha (Pelletier J P et al., “Cytokines and inflammation incartilage degradation” in Osteoarthritic Edition of Rheumatic DiseaseClinics of North America, Eds. R W Moskowitz, Philadelphia, W D.Saunders Company, 1993, p.545-568) and IL-1 receptors (Martel-Pelletieret al., Arthritis Rheum. 35: 530-540 (1992) have been found in diseasedjoints, and IL-1alpha induces cartilage matrix breakdown and inhibitssynthesis of new matrix molecules. Baragi et al., J. Clin. Invest. 96:2454-60 (1995); Baragi et al., Osteoarthritis Cartilage 5: 275-82(1997); Evans et al., J. Leukoc. Biol. 64: 55-61 (1998); Evans et al.,J. Rheumatol. 24: 2061-63 (1997); Kang et al., Biochem. Soc. Trans. 25:533-37 (1997); Kang et al., Osteoarthritis Cartilage 5: 139-43 (1997).Because of the association of IL-1alpha with disease, the WISPpolypeptide can also be assayed in the presence of IL-1alpha. Theability of the WISP polypeptide to not only have positive effects oncartilage, but also to counteract the catabolic effects of IL-1alpha isstrong evidence of the protective effect exhibited by the WISPpolypeptide. In addition, such and activity suggests that the WISPpolypeptide could inhibit the degradation which occurs in arthriticconditions, since catabolic events initiated by IL-1alpha are alsoinduced by many other cytokines and since antagonism of IL-1alphaactivity has been shown to reduce the progression of osteoarthritis.Arend, W. P. et al., Ann. Rev. Immunol. 16: 27-55 (1998).

The production of nitric oxide (NO) can be induced in cartilage bycatabolic cytokines such as IL-1. Palmer, R M J et al., Biochem.Biophys. Res. Commun. 193: 398-405 (1993). NO has also been implicatedin the joint destruction which occurs in arthritic conditions. Ashok etal., Curr. Opin. Rheum. 10: 263-268 (1998). Unlike normal (undiseased oruninjured) cartilage, osteoarthritic cartilage produced significantamounts of nitric oxide ex vivo, even in the absence of added stimulisuch as interleukin-1 or lipopolysaccharide (LPS). In vivo animal modelssuggest that inhibition of nitric oxide production reduces progressionof arthritis. Pelletier, J P et al., Arthritis Rheum. 7: 1275-86 (1998);van de Loo et al., Arthritis Rheum. 41: 634-46 (1998); Stichtenoth, D.O. and Frolich J. C., Br. J. Rheumatol. 37: 246-57 (1998). In vitro,nitric oxide exerts detrimental effects on chondrocyte function,including inhibition of collagen and proteoglycan synthesis, inhibitionof adhesion to the extracellular matrix, and enhancement of cell death(apoptosis). Higher concentrations of nitrite are found in synovialfluid from osteoarthritic patients than in fluid from rheumatoidarthritic patients. Renoux et al., Osteoarthritis Cartilage 4: 175-179(1996). Furthermore, animal models suggest that inhibition of nitricoxide production reduces progression of arthritis. Pelletier, J. P. etal., Arthritis Rheum. 7: 1275-86 (1998); van de Loo et al., ArthritisRheum. 41: 634-46 (1998); Stichtenoth, D. O. & Frolich, J. C., Br. J.Rheumatol. 37: 246-57 (1998). Since NO also has effects on other cells,the presence of NO within the articular joint could increasevasodilation and permeability, potentiate cytokine release byleukocytes, and stimulate angiogenic activity. Since NO likely play arole in both the erosive and the inflammatory components of jointdiseases, a factor which decreases nitric oxide production would likelybe beneficial for the treatment of degenerative cartilagenous disorders.

The assay to measure nitric oxide production is based on the principlethat 2,3-diaminonapthalene (DAN) reacts with nitrite under acidicconditions to form 1-(H)-naphthotriazole, a fluorescent product. As NOis quickly metabolized into nitrite (NO₂ ⁻¹) and nitrate (NO₃ ⁻¹),detection of nitrite is one means of detecting (albeit undercounting)the actual NO produced by cartilage.

The ability of a WISP polypeptide to enhance, promote or maintain theviability of chondrocytes in cultures in the absence of serum or othergrowth factors can also be examined. Articular chondrocytes are firstprepared by removal of the extracellular matrix and cultured in amonolayer, which is believed to approximate the latter stages ofcartilage disorders when the matrix has been depleted. The assay is acolorimetric assay that measures the metabolic activity of the culturedcells based on the ability of viable cells to cleave the yellowtetrazolium salt MTT to form purple formazan crystals. This cellularreduction reaction involves the pyridine nucleotide cofactors NADH andNADPH. Berridge, M. V. & Tan, A. S., Arch. Biochem. Biophys. 303: 474(1993). The solubilized product is spectrophotometrically quantitated onan ELISA reader.

Yet another assay examines the effects of WISP polypeptides onproteoglycan synthesis in patellae (kneecaps) of mice. This assay usesintact cartilage (including the underlying bone) and thus tests factorsunder conditions which approximate the in vivo environment of cartilage.Compounds are either added to patellae in vitro, or are injected intoknee joints in vivo prior to analysis of proteoglycan synthesis inpatellae ex vivo. As has been shown previously, in vivo treated patellaeshow distinct changes in PG synthesis ex vivo (Van den Berg et al.,Rheum. Int. 1: 165-9 (1982); Vershure, P. J. et al., Ann. Heum. Dis. 53:455-460 (1994); and Van de Loo et al., Arthrit. Rheum. 38: 164-172(1995). In this model, the contralateral joint of each animal can beused as a control.

A guinea pig model can be employed to measure the effects of WISPpolypeptides on both the stimulation of PG synthesis and inhibition ofPG release in articular cartilage explants from a strain of guinea pigs,Dunkin Hartley (DH), which spontaneously develops knee osteoarthritis(OA). Most other animal models which cause rapidly progressing jointbreakdown resemble secondary OA more than the slowly evolving humanprimary OA. In contrast, DH guinea pigs have naturally occurring slowlyprogressive, non-inflammatory OA-like changes. Because the highlyreproducible pattern of cartilage breakdown in these guinea pigs issimilar to that seen in the human disorder, the DH guinea pig is awell-accepted animal model for osteoarthritis. Young et al.,“Osteoarthritis”, Spontaneous animal models of human disease vol. 2, pp.257-261, Acad. Press, New York. (1979); Bendele et al., Arthritis Rheum.34: 1180-1184; Bendele et al., Arthritis Rheum. 31: 561-565 (1988);Jimenez et al., Laboratory Animal Sciences 47 (6): 598-601 (1997); Weiet al., Acta Orthop Scand 69: 351-357 (1998)). Initially, these animalsdevelop a mild OA that is detectable by the presence of minimalhistologic changes. However, the disease progresses, and by 16-18 monthsof age, moderate to severe cartilage degeneration within the joints isobserved. As a result, the effect of the WISP polypeptide on thecartilage matrix of the DH guinea pigs over the progression of thedisease would be indicative of the therapeutic effect of the compound inthe treatment of OA at different stages of joint destruction.

The metabolic changes associated with diabetes mellitus (diabetes)affect may other organ and musculo-skeletal systems of the afflictedorganism. For example, in humans, the incidence of musculoskeletalinjuries and disorders is increased with the onset of diabetes, anddiabetes is considered a risk factor for the development of arthritis.

A syndrome similar to diabetes can be induced in animals byadministration of streptozotocin (STZ). Portha B. et al., Diabete Metab.15: 61-75 (1989). By killing pancreatic cells which produce insulin, STZdecreases the amount of serum insulin in treated animals. STZ-induceddiabetes is associated with atrophy and depressed collagen content ofconnective tissues including skin, bone and cartilage. Craig, R. G. etal., Biochim. Biophys. Acta 1402: 250-260 (1998). In this assay, thepatellae of treated STZ-treated mice are incubated in the presence ofthe WISP polypeptide and the resulting matrix synthesis is analyzed. Theability of the WISP polypeptide to increase or restore the level of PGsynthesis to that of untreated controls is indicative of the therapeuticpotential.

In another embodiment of the invention, kits and articles of manufacturecontaining materials useful for the diagnosis or treatment of thedisorders described above are provided. The article of manufacturecomprises a container and an instruction. Suitable containers include,for example, bottles, vials, syringes, and test tubes. The containersmay be formed from a variety of materials such as glass or plastic. Thecontainer holds a composition which is effective for diagnosing ortreating the degenerative cartilagenous disorder, and may have a sterileaccess port (for example the container may be an intravenous solutionbag or a vial having a stopper pierceable by a hypodermic injectionneedle). The active agent in the composition will typically be a WISPpolypeptide. The composition can comprise any or multiple ingredientsdisclosed herein. The instruction on, or associated with, the containerindicates that the composition is used for diagnosing or treating thecondition of choice. For example, the instruction could indicate thatthe composition is effective for the treatment of osteoarthritisarthritis, rheumatoid arthritis or any other degenerative cartilagenousdisorder. The article of manufacture may further comprise a secondcontainer comprising a pharmaceutically-acceptable buffer, such asphosphate-buffered saline, Ringer's solution and dextrose solution.Alternatively, the composition may contain any of the carriers,excipients and/or stabilizers mentioned herein. It may further includeother materials desirable from a commercial and user standpoint,including other buffers, diluents, filters, needles, syringes, andpackage inserts with instructions for use.

The following examples are offered for illustrative purposes only, andare not intended to limit the scope of the present invention in any way.

All patent and literature references cited in the present specificationare hereby incorporated by reference in their entirety.

EXAMPLES

Commercially available reagents referred to in the examples were usedaccording to manufacturer's instructions unless otherwise indicated. Thesource of those cells identified in the following examples, andthroughout the specification, by ATCC accession numbers is the AmericanType Culture Collection, Manassas, Va. Unless otherwise noted, thepresent invention uses standard procedures of recombinant DNAtechnology, such as those described hereinabove and in the followingtextbooks: Sambrook et al., Molecular Cloning: A Laboratory Manual, ColdSpring Harbor Press N.Y., 1989, Ausubel et al., Current Protocols inMolecular Biology, Green Publishing Associates and Wiley Interscience,N.Y., 1989; Innis et al., PCR Protocols: A Guide to Methods andApplications, Academic Press, Inc., N.Y., 1990; Harlow et al.,Antibodies: A Laboratory Manual, Cold Spring Harbor Press, Cold SpringHarbor, 1988; Gait, M. J., Oligonucleotide Synthesis, IRL Press, Oxford,1984; R. I. Freshney, Animal Cell Culture, 1987; Coligan et al., CurrentProtocols in Immunology, 1991.

Example 1

In the assays described below, the following methods and materials wereemployed:

Materials: Chondroitin sulfate A from bovine trachea, chondroitinsulfate C from shark cartilage, hyaluronidase (EC 3.2.1.45) from bovinetestes and chondroitinase AC II (EC 4.2.2.5) from Artherobacteraurenscens were purchased from Calbiochem (San Diego). Chondroitinsulfate B, heparin and heparan sulfate from porcine intestinal mucosa,decorin and biglycan from bovine articular cartilage, chondroitinase C,chondroitinase B and heparinase I (EC 4.2.2.7) from Flavobacteriumhepanium were obtained from Sigma. Chondroitin sulfate D from sharkcartilage and chondroitin sulfate E from squid cartilage were purchasedfrom United States Biological (Swampscott, Mass.). Neuramimidase (EC3.2.2.18) from Vibrio Cholera, chondroitinase ABC (EC 4.2.2.4), proteasefree from Proteus vulgaris, Complete EDTA-free protease inhibitorscocktail tablets and fatty acid ultra free BSA fraction V were purchasedfrom Roche Molecular Biochemicals (Indianapolis, Ind.).Chondroitin-4-sulfatase (EC 3.1.6.9) and chondroitin-6-sulfatase (EC3.1.6.10) from Proteus vulgaris were from ICN Biomedicals (Aurora, OI).Horseradish peroxidase conjugated and biotinylated goat anti-human IgG,Fc fragment specific and biotinylated anti-sheep IgG was purchased fromJackson ImmunoResearch (Costa Mesa, Calif.). Proteinase K (EC 3.4.21.14)ready-to-use, Texas red conjugated steptavidin and anti-vimentinmonoclonal antibody (clone Vim 3B4) was from Dako (Carpinteria, Calif.).5-chloromethylfluoroscein diacetate (5-CFDA) and Hoechst 33342 werepurchased from Molecular Probes (Eugene, Oreg.). The Renaissance TSAindirect amplification kit was bought from NEN Life Science Products(Boston, Mass.). Vectashield mounting media and biotinylated horseanti-mouse IgG were obtained from Vector (Burlingame, Calif.).

Full length murine WISP-1 (Pennica et al., Proc. Natl. Acad. Sci.,95:14717-14722 (1998); WO 99/21998) was cloned into an expression vectorencoding the human IgG1 Fc region downstream of the WISP-1 sequence asdescribed previously for TNFR1 (Ashkenazi et al., Proc. Natl. Acad.Sci., 88:10535-10539 (1991)). The resulting recombinant fusion protein(WISP-1-Fc) was synthesized in a baculovirus expression system using Sf9insect cells and purified to homogeneity from serum-free conditionedmedium by affinity chromatography on a protein A-Sepharose Fast Flow(Pharmacia Biotech, Sweden) column. Unadsorbed proteins were washed outwith 50 mM sodium phosphate buffer containing 1 M NaCl. WISP-1-Fc waseluted with 100 mM glycine pH 2.5 and the pH was neutralized with 0.1volume of 3M Tris-HCl pH 8. After dialysis (20 mM Tris-HCl, pH 7.5, 150mM) the purified protein was concentrated by ultrafiltration usingCentriprep-30 (Millipore Corp., Bedford, Mass.) and the purity estimatedby SDS-PAGE and silver staining. The experiments were repeated at leastthree times with three different batches of protein expressed andpurified at different times and similar results were obtained.

Cell Culture: NRK (normal rat kidney fibroblasts), Hs 597.5k (humannormal skin fibroblasts), Hs 839.T (human skin melanoma fibroblasts), Hs908.5k (human skin melanoma fibroblasts), COLO 320DM (human colonadenocarcinoma cells), RAG (mouse renal adenocarcinoma cells), 293(human kidney epithelial cells), HUVEC (human umbilical vein endothelialcells), and WM-266-4 (human skin melanoma epithelial cells) wereobtained from American Type Culture Collection, Manassas, Va. The cellswere maintained in Low glucose Dulbecco's modified Eagle's Medium/HamF-12 (1:1) supplemented with 10% FBS at 37° C. under 5% CO₂.

Cell Binding: Cells were plated in 8 well plastic chamber slides andmaintained overnight at 37° C., 5% CO₂. The next day the cells werewashed with PBS and the wells were blocked for 30 minutes at roomtemperature with 3% BSA in HBS-C buffer (25 mM Hepes, pH 7.2, 150 NaCl,3 mM CaCl₂, 3 mM MgSO₄, 5 mM KCl, Complete protease inhibitorscocktail). When indicated, cells were washed and incubated 2 hours at37° C. with 0.1 U of the different lyases before blocking. (see,Vacherot et al., J. Biol. Chem., 274:7741-7747 (1999)). The cells wereincubated with 1 nM mWISP-1-IgG for 1 hour at room temperature, washedand incubated with 0.2 μg/ml biotinylated anti-human IgG Fc′ in HBS-C/3%BSA for 30 minutes at room temperature. The signal was amplified usingthe TSA indirect kit (NEN Dupont) according to the manufacturerinstructions. After a 30 minutes incubation with 1:200 FITC conjugatedstreptavidin (DAKO), the slides were mounted using Vectashieldcontaining 1 μg/ml Hoechst 33342 (Molecular Probes) and visualized undera Nikon Eclipse 800 fluorescent microscope. The images were acquiredusing a Photometrics 300 CCD Cooled Camera. Measurement of thefluorescence intensity of cells was as described previously withmodifications (Szurdoki et al., Anal. Biochem., 291:219-228 (2001)).Briefly, images of a minimum of three separate fields containing anaverage of 90 cells were acquired and stored as electronic files. Thethreshold was defined as the lowest intensity of the 1% brightest pixelsin a negative control executed without WISP-1-Fc. The fluorescencesignal for a cell population was defined as the total pixel intensityover the threshold divided by the cell number.

Solid Phase Binding Assay: Proteins were diluted in 50 μl (total volume)of PBS, applied to polystyrene microtiter wells and incubated at 40 Covernight. The next day the wells were washed three times with 300 μl ofHBS-c containing 0.3% BSA and the non-specific binding sites wereblocked for 1 hour at room temperature with 200 μl HBS-C/3% BSA. Thebuffer was aspirated and 50 μl of 0.5 nM WISP-1-IgG in HBS-C/3% BSA wasincubated for 2 hours at room temperature. The wells were washed andincubated for 1 hour with 50 μl of 2 μg/ml horseradish peroxidaseconjugated goat anti-human IgG Fc′ in HBS-C/3%. At the end of theincubation, the wells were washed 6 times with 200 μl of PBS containing0.05% Tween-20 and the signal was visualized using 100 μl of thehorseradish peroxidase chromogenic substrate TMB (KPL). The reaction wasstopped with 100 μl of 1 M phosphoric acid and the OD at 450 nm wasmeasured. Non-specific WISP-1-Fc binding was determined in parallelincubations by omitting microtiter well coating. No signal was generatedwhen WISP-1-Fc was omitted.

Purification of WISP-1 Binding Factors: Human skin fibroblasts werecycled between serum containing and serum free culture media every 3days. The serum free conditioned media was concentrated on aCentriprep-30 (Millipore, Bedford, Mass.). The buffer was then changedby sequentially adding 20 mM Tris-HCL pH 7.4, 300 mM NaCl andreconcentrating. The concentrate (150 μg protein/ml) was snap frozen andstored at −80° C. until used. The concentrated conditioned media wasthawed, filtered and applied on a Mono Q anion exchange columnequilibrated in 20 mM Tris-HCl pH 7.4, containing 300 mM NaCl. Thecolumn was washed and the adsorbed proteins were eluted using a lineargradient of NaCl (300 mM-2 M) in the same buffer. Fractions of 500 μlwere analyzed for WISP-1 binding activity.

Protein Identification by Mass Spectrometry: The fractions containingthe WISP-1 binding activity were pooled, denatured, reduced and appliedon 4-15% gradient acrylamide SDS-PAGE with or without a previousincubation for 2 hours at 37° C. with 0.1 U of chondroitinase ABC. Thegels were silver stained and the protein bands demonstrating a mobilitychange upon chondroitinase ABC digestion were excised and digested insitu with trypsin as previously described by Arnott et al.,Electrophoresis, 19:968-980 (1998). Tryptic peptides were extracted andanalyzed by microcapillary reverse-phase liquid chromatography-massspectrometry. Peptide mixtures were loaded onto 100 μm i.d., 10 cm longfused silica capillary columns packed with 5 μm C18 beads (238MSB5;Vydac, Hesperia, Calif.) and eluted with an acetonitrile gradientdirectly into the microelectrospray ionization source of an ion trapmass spectrometer (LCQ; Thermoquest, San Jose, Calif.). A flow rate of500 μl/min was obtained by a pre-column split from 25 μl/min deliveredby the HPLC (Ultra PlusII; Microtech Scientific, Sunnyvale, Calif.;Arnott et al., supra). Automated, data-dependent acquisition of massspectra provided molecular mass (MS) and sequence data (MS/MS) forpeptides as they eluted from the column. Proteins were identified bycorrelation of MS/MS data with entries in a non-redundant proteinsequence database using the Sequest program (Gatlin, C., Eng, J., Cross,S., Detter, J. and Yates III, Analytical Chemistry, 72:757-763 (2000)).Protein matches were confirmed by manual interpretation of the spectra.

Immunofluorescence: Slide-mounted human colon tumor sections werebrought to room temperature, fixed with 70% ethanol for 10 minutes andthe non-specific binding sites were saturated with PBS/3% BSA containing1.5% normal serum for 20 minutes. The sections were incubated for 1 hourwith 0.125 microgram/ml anti-vimentin antibody, washed with PBS andfurther incubated for 30 minutes with 2 microgram/ml biotinylatedanti-mouse IgG antibody. The signal was amplified using the TSA indirectkit according to manufacturer instructions. After a 30 minute incubationwith 1:1000 Texas red conjugated streptavidin, the slides were mountedusing Vectashield containing 1 microgram/ml Hoechst 33342 and visualizedunder a Nikon Eclipse 800 fluorescent microscope. Images were acquiredusing a Photometrics 300 CCD Cooled Camera. The negative controlexecuted in absence of primary antibody did not reveal any fluorescentstaining.

The immunofluorescent detection of decorin on human skin fibroblasts wasexecuted using a similar protocol. 8×10³ cells were plated in chamberslides and cultured overnight. The next day, the cells were washed andincubated at 37° C. for 15 minutes with fresh medium containing 5microgram/ml 5-CFDA. After washing, the non-specific binding sites weresaturated with HBS-C/3% BSA for 30 minutes at room temperature. Thecells were then incubated for 1 hour at room temperature with 1:4000sheep anti-human decorin antibody in HBS-C/0.1% BSA. The cells werewashed, fixed with 4% paraformaldehyde/PBS for 10 minutes, washed andfurther incubated for 30 minutes with 2 microgram/ml biotinylatedanti-sheep IgG. The signal was amplified using the TSA indirect kit.After a 30 minute incubation with 1:1000 Texas red conjugatedstreptavidin, the slides were mounted using Vectashield containing 1microgram/ml Hoechst 33342 and visualized under a Nikon Eclipse 800fluorescent microscope. Images were acquired using a Photometrics 300CCD Cooled Camera. The negative control executed in absence of primaryantibody did not reveal any fluorescent staining.

Analytical Methods: SDS-PAGE was performed according to Laemli, Nature,227:680-685 (1970) using a Bio-Rad Mini-PROTEAN II vertical slab gelelectrophoresis apparatus. The apparent molecular mass was determinedusing the broad range molecular weight standards from Bio-Rad. Proteinwas determined using the Bio-Rad Protein Assay silver stain Dye Reagentand bovine serum albumin standard.

A. Binding of WISP-1 to Various Cell Lines and Human Colon TumorSections

The binding of a chimeric recombinant mouse WISP-1 bearing a humanimmunoglobulin Fc fragment tag to various cells in culture was analyzed.Cells were seeded in chamber slides and cultured overnight. The nextday, the non-specific binding sites were blocked and the cells wereincubated with 1 nM of mWISP-1-IgG or without mWISP-1-IgG for 1 hour.The cells were washed, fixed and the binding of WISP-1-IgG was detectedby immunofluorescence using a biotinylated anti-human IgG antibody andthe indirect tyramide substrate amplification procedure followed withFITC conjugated streptavidin.

As summarized in FIG. 1, the binding of WISP-1 could only be seen at thesurface of fibroblastic cell lines. As an example, the binding of WISP-1to NRK cells is illustrated (FIG. 1A). Moreover, the protein also boundto fibroblasts of rat or human origin whether they were normal or fromskin melanoma. On the other hand, no fluorescent signal could bedetected when mouse renal adenocarcinoma, human colon adenocarcinoma,human kidney epithelial cells, human umbilical vein endothelial cells,or human skin melanoma epithelial cells were used. As an example, thebinding of WISP-1 to RAG cells is illustrated (FIG. 1B). No signal couldbe detected when the addition of WISP-1 was omitted or when an unrelatedbiotinylated secondary antibody was used (FIG. 1C).

Binding of WISP-1 to human colon tumor sections was evaluated using insitu ligand binding procedures. Slide mounted human colon tumor sectionswere brought to room temperature and immediately incubated for 4 minutesin 35 mM acetic acid (pH 3.5) containing 3 mM CaCl₂, 3 mM MgSO₄, 5 mMKCl and 1 M NaCl. The slides were then washed in HBS-C (25 mM Hepes, pH7.2, 150 NaCl, 3 mM CaCl₂, 3 mM MgSO₄, 5 mM KCl, Complete proteaseinhibitor cocktail) containing 32 mM sucrose and the non-specificbinding sites were blocked for 20 minutes in HBS-C containing 3% BSA,1.5% normal goat serum and 32 mM sucrose. The binding sites were avidinand biotin were blocked using the avidin/biotin blocking kit from Vector(Burlingame, Calif.). The slides were incubated for 1 hour in HBS-C/3%BSA and 1 nM of WISP-1-Fc, washed three times for 1 minute each timewith cold (4° C.) HBS-C/1% BSA and fixed for 10 minutes in PBS/4%paraformaldehyde. The slides were incubated with 0.2 microgram/mlbiotinylated goat anti-human IgG, Fc specific in HBS-C/3% BSA for 30minutes, washed and fixed in PBS/4% paraformaldehyde for 10 minutes. Thesignal was amplified using the TSA indirect amplification kit accordingto the manufacturer instructions. The reaction was stopped by threewashes of 4 minutes in TBS/0.1% BSA. The slides were incubated for 30minutes with streptavidin conjugated FITC (1:1000) in TBS/0.1% BSA andwashed in TBS containing 0.05% Tween-20. The sections were mounted usingVectashield mounting media containing 1 microgram/ml Hoechst 33342 andvisualized under a Nikon Eclipse 800 Fluorescent microscope.

Although vimentin staining revealed the presence of mesenchymal cells inboth the tumor and the normal mucosa (see FIGS. 1F and 1G), the in situWISP-1 binding was restricted to the peritumoral stroma (FIG. 1D). Nobinding was found to the tumor epithelial cells or to the normal mucosa(FIGS. 1D and 1E).

B. WISP-1 Binds to Human Skin Fibroblast Conditioned Media

To examine whether a WISP-1 binding factor was secreted or shed from thesurface of human skin fibroblasts, a solid phase binding assay wasconducted. Serum free conditioned media from human skin fibroblasts(prepared as described above) was collected, concentrated and coated inmicrotiter plates overnight. Fifty microliters of conditioned media wascoated in duplicate in microtitration wells. The non-specific bindingsites were saturated by incubation with HBS-C containing 3% BSA and thewells were incubated for 2 hours with mWISP-1-IgG. After blocking thenon-specific binding sites, the wells were first incubated with WISP-1and then with a horseradish peroxidase conjugated anti-human IgGantibody. The wells were washed and incubated for 1 hour withhorseradish peroxidase conjugated anti-human IgG Fc′. After 6 washeswith HBS-C containing 0.3% BSA, the signal was visualized using ahorseradish peroxidase chromogenic substrate. The reaction was stoppedwith 1 M phosphoric acid and the OD at 450 nm was measured. FIG. 2Ashows binding of 1 nM of mWISP-1-IgG to wells coated with serialdilutions of conditioned media, and FIG. 2B shows binding of serialdilutions of mWISP-1-IgG to wells coated with 0.5 μl of human skinfibroblast conditioned media. Binding was proportional to the amount ofmedia coated and the concentration of WISP-1 added, indicating thathuman skin fibroblasts produce soluble WISP-1 binding factors.

As seen in FIG. 3A, the interaction between WISP-1 and the conditionedmedia was abolished in the presence of 1 M NaCl. The presence of 100 mMEDTA only partially diminished the binding while the presence of 0.05%Tween-20 had no effect. It was concluded that the binding of WISP-1 tothe coated material was cation independent and had an ionic component.The possibility that the binding factor was a proteoglycan was theninvestigated by treating the coated wells with various lyases before thebinding of WISP-1 was evaluated. Treatment of the coated material withchondroitinase C, chondroitin-6-sulfatase, heparinase or neuramimidasedid not alter the binding of WISP-1 when compared to the control (FIG.3B). However, the digestion with chondroitinase AC II or hyaluronidasepartially diminished the binding. Ultimately, the treatment withchondroitinase ABC, chondroitinase B, chondroitin-4-sulfatase orproteinase K abolished the binding of WISP-1 to the coated wells. Thespecificity of chondroitinase B and chondroitin-4-sulfatase indicatesthat dermatan sulfate components are essential to the binding of WISP-1.Moreover, the sensitivity of the interaction to a proteinase K indicatesthat the binding factor has a proteinous component. The results suggestthat WISP-1 binds to a secreted dermatan sulfate containingproteoglycan.

Chondroitinase ABC and chondroitinase B treatments completely abolishedthe binding, whereas treatment with chondroitinase C had no effect.Chondroitinase B cleaves dermatan sulfate at thebeta-D-galactosamine-L-iduronic acid linkage. The specificity of thisenzyme demonstrates the requirement for iduronic acid for the binding ofWISP-1. Treatments with chondroitinase AC II or hyaluronidase onlypartially reduced the binding. This could indicate that theglycosaminoglycan chain responsible for the interaction of WISP-1consisted of a dermatan sulfate-chondroitin sulfate co-polymer. Bycleaving the susceptible galactosaminidic bonds, those enzymes couldhave removed parts of the glycosaminoglycan chain containing iduronicacid residues. Treatment with chondroitin-4-sulfatase completelyabolished the binding while chondroitin-6-sulfatase did not alter theinteraction. This indicates the necessity for a sulfate group atposition 4 of the N-acetylgalactosamine for the interaction. Treatmentwith heparinase had no effect, indicating that the binding does notrequire the iduronic acid to be sulfated at position 2. Treatment withproteinase K abolished the binding suggesting that the glycosaminoglycanresponsible for the interaction is linked to a protein core that couldbe detached from the wells by proteolytic degradation. Collectively,these results support the conclusion that a iduronic acid containingmotif of the glycosaminoglycan chain of a proteoglycan mediates WISP-1binding to human skin fibroblast conditioned media.

C. Purification and Identification of the WISP-1 Binding Factor

To purify the factor responsible for the binding of WISP-1, the serumfree conditioned media from human skin fibroblasts was collected afterthree days of culture, concentrated, transferred to a buffer containing20 mM Tris-HCl pH 7.4 300 mM NaCl and applied on a Q-Sepharose anionexchange chromatography column. The column was washed and the retainedproteins were desorbed with an increasing concentration of NaCl. Thepresence of a WISP-1 binding factor was analyzed in each fraction usinga solid phase binding assay, and the results are shown in FIG. 4A.Further, fraction 15 (indicated by a * in FIG. 4A) was incubated at 37°C. for 2 hours in the presence (+) or the absence (−) of 0.1 U ofchondroitinase ABC. The samples were separated by SDS-PAGE underreducing conditions and the gels were silver stained. The indicatedbands were identified by mass spectroscopy (FIG. 4B).

The bands found at 46, 60, and 70 kDa corresponded to decorin while theband at 44 kDa was identified as biglycan (the band at 230 kDa appearedto be a mixture of both decorin and biglycan). The bands found at thedifferent molecular weights probably corresponded to biglycan anddecorin containing incompletely digested glycosaminoglycan chains thatwere generated during the chondroitinase ABC treatment. The resultsdemonstrate that WISP-1 binds to the two dermatan sulfates containingproteoglycans, biglycan and decorin.

D. WISP-1 Binds to Decorin and Biglycan

To demonstrate the direct interaction of WISP-1 with decorin andbiglycan, a solid phase binding assay was conducted. Decorin andbiglycan were coated to microtiter wells overnight. Non-specific bindingsites were saturated and 0.25 nM of mWISP-1-IgG was incubated for 2hours. The wells were washed and incubated with horseradish peroxidaseconjugated anti-human IgG Fc′ (2 μg/ml) for 1 hour. After 6 washes withPBS containing 0.05% Tween-20, a signal was developed by the incubationof a chromogenic substrate. The color development was stopped by theaddition of 1 M phosphoric acid and the O.D. at 450 nm was measured.

As illustrated in FIG. 5A, the curves corresponding to the binding ofWISP-1 to decorin and biglycan are very similar and are proportional tothe amount of protein coated. Similarly, the ability of decorin andbiglycan to inhibit the binding of WISP-1 to coated human skinfibroblast conditioned media was evaluated.

Fifty microliters of human skin fibroblast conditioned media were coatedin wells of microtiter plates. Non-specific binding sites were saturatedand 0.25 nM of WISP-1-IgG was incubated in the presence of variousconcentrations of decorin (filled circles) or biglycan (empty circles)(FIG. 5B) for 2 hours. The binding of mWISP-1-IgG was evaluated asdescribed above. As seen in FIG. 5B, the binding of WISP-1 to the humanskin fibroblast conditioned media is gradually decreased in the presenceof increasing concentrations of decorin and biglycan. Decorin andbiglycan gave similar competition curves showing 50% inhibition of theWISP-1 binding at 70 μg/ml for decorin and 105 μg/ml for biglycan.

E. WISP-1 binds to Glycosaminoglycan

To understand if the specificity of the interaction of WISP-1 to theproteoglycan is limited to dermatan sulfate, the binding of WISP-1 tothe human skin fibroblast conditioned media in the presence of variousproteoglycans was evaluated. Serum free conditioned media of human skinfibroblasts was prepared as described above. Fifty μl of conditionedmedia were coated in wells of microplates overnight at 4° C., the nonspecific binding sites were saturated and the wells were incubated for 2hours at room temperature with 0.5 nM of WISP-1-IgG in the presence ofvarious concentrations of different glycosaminoglycans. The wells werewashed, a signal was developed using a chromogenic substrate and theO.D. at 450 nm was measured. FIG. 6 shows: Chondroitin sulfate A (filledcircles); dermatan sulfate (empty circles); chondroitin sulfate C(filled triangles); chondroitin sulfate D (empty triangles); chondroitinsulfate E (filled squares); heparin (X); heparan sulfate (emptysquares).

As shown in FIG. 6, the binding of WISP-1 is reduced proportionally inthe presence of increasing concentrations of various proteoglycans. Thebinding of WISP-1 reached 50% of the maximal binding at 3 μg/ml ofdermatan sulfate, 10.5 μg/ml chondroitin sulfate D or heparin, 30 μg/mlchondroitin sulfate E, 75 μg/ml of heparan sulfate, 105 μg/mlchondroitin sulfate A. The presence of chondroitin sulfate C did notreduce the binding of WISP-1. This data demonstrate that the interactionof WISP-1 with glycosaminoglycan is sufficient to mediate its binding tohuman skin fibroblasts conditioned media. Moreover it indicates thatWISP-1 shows a greater specificity for dermatan sulfate than any otherglycosaminoglycan tested.

F. Binding of WISP-1 to Human Skin Fibroblasts is Inhibited by DermatanSulfate

To ascertain the importance of dermatan sulfate containing proteoglycansin the binding of WISP-1 to the cell surface a cell binding analysis inthe presence of various glycoaminoglycans was performed. Human skinfibroblasts were seeded in chamber slides. The non specific bindingsites were saturated and 1 nM WISP-1-IgG was incubated for 1 hour atroom temperature in the absence (FIG. 7A) or the presence of 50 μg/mlchondroitin sulfate A (FIG. 7B), dermatan sulfate (FIG. 7C), chondroitinsulfate C (FIG. 7D), chondroitin sulfate D (FIG. 7E), chondroitinsulfate E (FIG. 7F), heparin (FIG. 7G) or heparan sulfate (FIG. 7H). Thecells were washed and fixed and the binding of WISP-1-IgG was detectedby immunofluorescence using a biotinylated anti-human IgG antibody andthe indirect tyramide substrate amplification procedure ended with FITCconjugated streptavidin.

In the absence of any added glycosaminoglycan the binding of WISP-1 tothe cell surface gave rise to a strong fluorescent staining. Chondroitinsulfate C and chondroitin sulfate D reduced WISP-1 binding byapproximately 20% and 46%, respectively, while chondroitin sulfate A,chondroitin sulfate E, heparin sulfate or heparin diminished theinteraction by approximately 60-70% (FIG. 7I). On the other hand, in thepresence of 50 μg/ml of dermatan sulfate, the binding of WISP-1 to thesurface of human skin fibroblasts was abolished. Together these resultsdemonstrate that WISP-1 has a higher affinity for dermatan sulfate andthis interaction may be responsible for the binding of WISP-1 to thecell surface.

G. WISP-1 Binding to Human Skin Fibroblasts is Abolished by theDigestion of the Cell Surface with Chondroitinase B

While WISP-1 interacts with glycosaminoglycans and small proteoglycanscontaining dermatan sulfate, whether it interacts with the cell surfacethrough the same type of interaction remained to be determined. Toaddress this possibility, the binding of WISP-1 to the surface of humanskin fibroblasts treated with different glycosaminoglycan lyases wasanalyzed. Human skin fibroblasts were incubated for 2 hours at 37° C. inthe absence (FIG. 8A), or the presence of with 0.1 U of chondroitinaseABC (FIG. 8B), chondroitinase B (FIG. 8C), chondroitinase C (FIG. 8D),heparinase (FIG. 8E), or in the absence of mWISP-1 (FIG. 8F). The cellswere washed, the non specific binding sites were saturated and 1 nMmWISP-1-IgG was incubated for 1 hour at room temperature. After 3washes, the cells were fixed and the binding of mWISP-1-IgG was detectedby immunofluorescence using a biotinylated anti-human IgG antibody andthe indirect tyramide substrate amplification procedure ended with FITCconjugated streptavidin.

As shown in FIG. 8A, the binding of WISP-1 to the surface of untreatedhuman skin fibroblasts gave rise to a strong fluorescent signal. Whenthe cells were treated with chondroitinase ABC or chondroitinase B thebinding of WISP-1 was decreased to a level comparable to the negativecontrol in which WISP-1 was omitted (FIGS. 8B, C and D respectively). Onthe other hand, the binding of WISP-1 to the cells treated withchondroitinase C or heparinase did not show any modification in term ofdistribution or intensity (FIG. 8, panel D and E respectively). Theseresults indicated that the binding of WISP-1 to the cell surface ofhuman skin fibroblasts is mediated by a dermatan sulfate containingproteoglycan.

H. Decorin and Biglycan Block the Binding of WISP-1 Human SkinFibroblasts

The binding of WISP-1 to human skin fibroblasts was evaluated in thepresence or the absence of an excess of decorin or biglycan. Human skinfibroblasts were seeded in chamber slides and the non specific bindingsites were saturated. One nanomolar mWISP-1-IgG was incubated for 1 hourat room temperature in the presence of 1 mg/ml decorin (FIG. 9A),biglycan (FIG. 9B), or in the absence of added competitors (FIG. 9C).The cells were washed and fixed, and the binding of WISP-1-IgG wasdetected by immunofluorescence using a biotinylated anti-human IgGantibody and the indirect tyramide substrate amplification procedureended with FITC conjugated streptavidin.

As shown in FIG. 9, the presence of decorin or biglycan partiallyblocked the interaction of WISP-1 with human skin fibroblasts. Althoughthe inhibition is significant (approximately 88% and 94%), even at thehighest concentration tested (1 mg/ml) the binding could not becompletely abolished. This can be explained by the capacity that decorinand biglycan have to interact with collagen present in the extracellularmatrix of the cell.

Decorin and biglycan are members of a family of small leucine-richproteoglycans present in the extracellular matrix of connective tissues.The secreted form of decorin consists of a core protein of 36,319 Da(Krusius et al., Proc. Natl. Acad. Sci., 83:7683-7687 (1986)) and asingle glycosaminoglycan chain of dermatan sulfate attached to a serineat position 4 (Scott, PG, Dermatan Sulfate Proteoglycans: Chemistry,Biology, Chemical Pathology, Portoland Press, London, England, 1993).The secreted form of biglycan consists of a core protein of 37,983 Dasubstituted with two glycosaminoglycan chains, one of dermatan sulfateand one of chondroitin sulfate (Fisher et al., J. Biol. Chem.,264:4571-4576 (1989)). The core proteins of biglycan and decorin shareabout 55% amino acid identity. The molecular weight of the core proteinof decorin and biglycan corresponds to the predicted molecular weight ofthe two bands referred to above having the fastest electrophoreticmobility after the chondroitinase ABC treatment. The slower migratingbands would correspond to decorin and biglycan bearing partiallydigested glycosaminoglycan chains.

Decorin co-localizes with fibronectin fibrils at the surface of humanskin fibroblasts (Schmidt, G., Robenek, H., Harrach, B., Glossl, J.,Nolte, V., Hormann, H., Richter, H. and Kresse, H., J. Cell. Biol,104:1683-1691 (1987)). It is possible that the WISP-1 interaction withthe cell surface is mediated by decorin attached to the extracellularmatrix. Using immunofluorescence, the presence of decorin at the surfaceof the human skin fibroblasts was confirmed in the above assays. Also,it was shown that decorin and biglycan significantly diminished WISP-1binding to the cell surface. The interaction of decorin and biglycanwith human skin fibroblasts probably prevented the complete inhibitionof WISP-1 binding Together those results demonstrated that decorin canact as a cell surface binding site for WISP-1.

Several proteoglycans associated with the cellular membrane or theextracellular matrix were shown to contain iduronic acid. Consequently,it is possible for WISP-1 to interact with chondroitin sulfate ofheparan sulfate proteoglycan exhibiting iduronate motifs. Also, theiduronic acid content of the glycosaminoglycan chain of differentproteoglycans was shown to vary with their tissue distribution. Forexample, decorin and biglycan from skin contain approximately 80% ofiduronic acid whereas in cartilage they contain only 40% (Choi et al.,J. Biol. Chem., 264:2876-2884 (1989)). The glycosaminoglycan chains ofbiglycan and decorin from bone and bovine nasal cartilage contain noiduronate and are therefore chondroitin sulfate (Fisher et al., J. Biol.Chem. 262:9702-9708 (1987); Heinegard et al., Biochem. J., 3:2042-2051(1981)). Also, it was reported that TGF-β treatment induces a 10 to 15%decrease of the iduronic acid content of side-chains of decorin andbiglycan (Malmstrom, A et al., Dermatan Sulfate Proteoglycans:Chemistry, Biology, Chemical Pathology, Portoland Press, London,England, 1993). Consequently, it is possible that modification in thelevel of iduronic acid content in the glycosaminoglycan chain ofproteoglycans modulates the interaction of WISP-1.

Biglycan and decorin are known to interact with a variety ofextracellular matrix proteins, cytokines and cell surface receptors (fora review, see Hocking et al., Matrix Biol., 17:1-19 (1998) and Iozzo, R.V. J. Biol. Chem. 274:18843-18846 (1999). Decorin and biglycan interactwith transforming growth factor-β (TGF-β), negatively regulating itsbiological activity (Hildebrand et al., Biochem. J., 302:527-534(1994)). Also, decorin was shown to decrease mRNA levels and TGF-βprotein synthesis in vitro (Stander et al., Gene Therapy, 5:1187-1194(1998)). On the other hand, the expression of decorin is generallydownregulated by TGF-β in various cells and organisms (Iozzo, Ann. Rev.Biochem., 67:609-652 (1998)). The promoter region of the decorin genecontains a TGF-β-negative regulated element. This TGF-β-negativeregulated element has been found in several protease genes downregulatedby TGF-β and could function to suppress the decorin gene expression(Iozzo, Experientia, 49:447-455 (1993)). Moreover, the expression ofdecorin well correlates with a malignant property in human carcinoma(Adany et al., J. Biol. Chem., 265:11389-11396 (1990); Hunzlemann etal., J. Invest. Sermatol., 104:509-513 (1995)). It was found to bedepressed in many tumoral tissues (Iozzo, supra, 1993) and lost inseveral tumor cell lines (Iozzo et al., FASEB J., 10:598-614 (1996)).However, the expression of decorin is increased in the tumoral stroma(Adany et al., supra, 1991; Iozzo, supra, 1993, Brown et al., Clin.Cancer Res., 5:1041-1056 (1999)). Decorin could be a potent negativeregulator of the TGF-β released by the tumor to facilitatecarcinogenesis and tumor progression. Since decorin was shown todirectly suppress the growth of several carcinomas through TGF-βdependent and independent mechanisms, it was proposed that itsexpression in the peritumorous stroma may reflect a regional response ofthe host connective tissue cells to the invading neoplastic cells(Stander et al., supra, 1999).

Example 2 Adhesion of CHO Cells to WISP-1 and Other ECM Proteins

The following CHO cell lines (identified by ATCC number) were maintainedin Ham-F12/LGDMEM (50:50) containing 10% FBS:

CHO-K1 (CCL-61) CHO pgs A-745 (CRL-2242; DO NOT synthesize proteoglycan)CHO pgs B-618 (CRL-2241; DO NOT synthesize proteoglycan) CHO pgs D-677(CRL-2244; DO NOT synthesize haparan sulfate) CHO pgs E-606 (CRL-2246;Synthesize an undersulfated heparan sulfate)Maxisorp plates were coated with 50 μl of mWISP-1-IgG (5 μg/ml) or BSA3% (Fraction V, fatty acid ultra-free; Boehringer Mannheim) in solutionin PBS at 4° C. overnight. The next day, the contents of the wells wereaspirated and the wells blocked with 200 μl of PBS/3% BSA for 1 hour atroom temperature. The cells were taken up in PBS containing 2 mM EDTA,and the clumps were broken using a pipette and then centrifuged at 1000rpm for 10 minutes. The supernatant was removed, and the cells werewashed twice with serum free Ham-F12/LGDMEM (50:50) containing 1% BSA.

The cells were resuspended at 25×10⁵ cells/ml in serum freeHam-F12/LGDMEM (50:50) containing 1% BSA. 50 μl of serum freeHam-F12/LGDMEM (50:50)/1% BSA was added to each well followed by 50 μlof cell suspension. The plates were incubated 2 hours at 37° C. withoutlid. Subsequently, the wells were washed 3× with PBS and once thesupernatant was completely removed, the plates were stored at −70° C.

The plates were thawed and Molecular Probes CyQUANT (Molecular Probes)was added. Fluorescence was measured at 480 nm-520 nm.

The results are shown in FIG. 10.

Mutant CHO cell lines impaired in their glycosaminoglycan synthesis wereused to verify the role of the proteoglycan in the cell adhesion toWISP-1. As shown in FIG. 10, none of the CHO cell lines completelydeficient for the synthesis of glycosaminoglycan (CHO pgs A and CHO pgsB) were found to adhere to WISP-1. This result indicates that theadhesion of CHO cells to WISP-1 is totally dependent on theglycosaminoglycan side chains of the proteoglycan. On the other hand,CHO cell lines lacking heparan sulfate (CHO pgsD) or synthesizing anundersulfated heparan sulfate showed a 40% reduction in adhesion toWISP-1 compared to CHO-K1 that synthesize a normal proteoglycan. Thisshows that heparan sulfate proteoglycan of CHO cells is responsible onlyin part for the cell adhesion to WISP-1 and that its sulfation isnecessary for its activity. Consequently, the dermatan sulfateproteoglycan which is the remaining fraction of the proteoglycan of CHOpgs D and CHO pgs E should be responsible for most of the adhesion ofCHO cells to WISP-1.

Example 3 Adhesion of Human Skin Fibroblasts to WISP-1 and Other ECMProteins

Human Skin Fibroblasts (ATCC; CRL 7356) were maintained inHam-F12/LGDMEM (50:50) containing 10% FBS. Maxisorp plates were coatedwith 50 μl of the proteins (identified below) in solution in PBS at 4°C. overnight:

Collagen I, Human (2 μg/ml) (Human; BioDesign) Collagen II, Human (2μg/ml) (Human; BioDesign) mWISP-1-IgG (2 μg/ml) (see Example 1 above)BSA 3% (Fraction V, fatty acid ultra-free; Boehringer Mannheim)The next day, the content of the wells was aspirated and the wells besaturated with 200 μl of PBS/3% BSA for 1 hour at room temperature. Thecells were taken up in PBS containing 15 mM EDTA, and the clumps werebroken up using a pipette. The cell suspension was filtered over a 45 μmfilter and centrifuged at 1000 rpm for 10 minutes.

The supernatant was removed and the cells washed twice with serum freeHam-F12/LGDMEM (50:50) containing 1% BSA. The cells were resuspended at3×10⁵ cells/ml with serum free Ham-F12/LGDMEM (50:50) containing 1% BSA.50 μl of serum free Ham-F12/LGDMEM (50:50)/1% BSA was then added, alongwith 100 μg/ml Dermatan sulfate (Chondroitin sulfate B from Porcineintestinal mucosa; Sigma); 100 μg/ml Heparin (Porcine intestinal mucosa;Sigma) or no addition. The plates were incubated for 15 minutes at roomtemperature. Then, 50 μl of cell suspension was added to each well andincubated 2 hours at 37° C. without lids. Subsequently, the wells werewashed 3× with PBS. Staining was performed with crystal violet for 30minutes. The plates were then washed in water. O.D. was measured at 570nm.

The results are shown in FIG. 11.

The data suggests that although the value is lower than the positivecontrols (adhesion to collagen I and to collagen II) human skinfibroblasts adhere to wells coated with WISP-1 (FIG. 11). The presenceof 100 μg/ml of heparin or 100 μg/ml dermatan sulfate reduced the celladhesion to WISP-1 by 30% or 70% respectively. In similar conditions,the cell adhesion to collagen I and II did not significantly change.Those results indicate that the adhesion of human skin fibroblasts toWISP-1 is mediated through a different mechanism than the adhesion tocollagen I and collagen II. It also indicates that while heparincontaining proteoglycan can participate in this phenomenon, the adhesionof the human skin fibroblasts to WISP-1 is mainly mediated throughdermatan sulfate proteoglycan.

Example 4 Chondrocyte Re-Differentiation Assay

An experiment was conducted to determine the effects of variousconcentrations of WISP-1 polypeptides on chondrocyte differentiation. Inorder to culture chondrocytes, articular cartilage is digested withenzymes which remove the extracellular matrix. Thus, the cellularenvironment in this culture system may be similar to that found in laterstages of cartilage disorders where the matrix has been depleted andchondrocyte cells tend to revert back to an “immature” phenotype.

The metacarpophalangeal joints of 4-6 month old female pigs wereaseptically dissected, and articular cartilage was removed by free-handslicing taking care so as to avoid the underlying bone. These cartilagefragments were then digested in 0.05% trypsin in serum-free Ham's F12for 25 minutes at 37° C. The medium was drained and discarded, andcartilage was digested in 0.3% collagenase B in serum-free Ham's F12media for thirty minutes at 37° C. The medium was drained and discarded,and the cartilage was digested overnight in 0.06% collagenase B in Ham'sF12+10% fetal bovine serum. The cells were then filtered through a 70micron nylon filter and seeded in Ham's F12 medium without serum. Theisolated cells are seeded at 25,000 cells/cm² in Ham F-12 containing 10%FBS and 4 μg/ml gentamycin. The culture media was changed every thirdday and the cells were re-seeded to 25,000 cells/cm² every five days. Onday 12, the cells were seeded in 96 well plates at 5,000 cells/well in100 μl of the same media without serum and 100 μl of human WISP-1-IgG(see FIG. 17), at 1% dilution, were added to give a final volume of 200μl/well. After 5 days at 37° a picture of each well was taken using astage driven inverted microscope. The differentiation state wasmorphologically determined using the Metamorph software from UniversalImaging Corporation. On each picture, round cells corresponding tore-differentiated chondrocytes are selected according to their size andshape whereas flattened cells having a dedifferentiated phenotype areeliminated. The total area covered by the selected cells on a picture isthen calculated and compared to a positive control (redifferentiatedchondrocytes by Staurosporin) and to a negative control (untreatedcells).

The result calculation and interpretation were conducted as follows:

Result Calculation:Y=Chondrocytes areaRedifferentiationindex=[(Y−Y_(negative control))/(Y_(positive control)−Y_(negative control))]*100Result Interpretation:

The greater the redifferentiation index, the better the WISP moleculewill promote the chondrocytes to redifferentiate.

Result Cutoff: Redifferentiation index>40→positive result.

The results are shown in FIG. 12.

Example 5 Collagen II Staining Assay

Collagen II is a preferred marker for chondrocytes. After primaryporcine chondrocytes are in culture for 10 days to “de-differentiate”into mesenchymal cells, the cells tend to loose their collagen IIexpression. The chondrocyte differentiation assay described above wasconducted in which triplicate wells were treated for (+) and (−)controls and duplicates of each of the following proteins for 5 days:

Positives  5 nM (0.5 μl/50 ml) Staurosporin 100 nM IGF-1 Negative mediumalone Test 100 nM human WISP-1-His 100 nM human WISP-2-His 100 nM humanWISP-3-His

(The WISP polypeptide constructs were prepared using a N-terminal Histag attached to each WISP polypeptide).

After the pictures are taken using the inverted microscope (as describedin Example 4), the cells were fixed in 70% ethyl alcohol for 15 minutesat room temperature, and then washed 3× with PBS. The plates wereblocked with PBS/3% BSA for 60 minutes at 200 μl/well. The treated wellswere treated with mouse anti-Collagen II (Neomarker-5 B2.5) in PBS/3%BSA for 1 hour at room temperature, running a 1:2000 dilution. Theplates were again washed 3× with PBS/0.1% BSA.

Following the wash, the plates were incubated with 1:1000 of VectorBiotinylated anti-Mouse in PBS/0.1% BSA for 30 minutes at roomtemperature. The plates were then washed 3× for 2 minutes in PBS.

The cells were fixed in 4% paraformaldehyde in PBS for 10 minutes atroom temperature, and then washed with TBS (50 mM Tris-HCl, 150 mM NaCl,pH 8)+0.3%. BSA, 2× for 3 minutes (500 μl/well). Then, incubate withDupont HRP-Streptavidin 1:1000 in TBS+1% BSA for 30 minutes (100μl/well). This is followed by washes with TBS+0.1% BSA, 3× for 4 minutes(500 μl/well). Next, incubate with biotinylated tyramide for 10 minutes1:50 in amplification diluent (NEN Dupont) (100 μl/well). Washes withTBS+0.1% BSA, 3× for 4 minutes (500 μl/well) followed the incubation.

In the next incubation, DAKO FITC-Streptavidin 1:1000 in TBS in HBS-C+1%BSA was added for 30 minutes (100 μl/well). Then, there was a brief washin PBS. Finally, 1:1000 Hoechst in PBS (100 μl/well) was added, and thenevaluated under an inverted microscope.

The data are shown in FIG. 13. Positive controls (Staurosporin andIGF-1) stained strongly for collagen II while the negative control didnot show any staining at all. Cells treated with 100 nM of WISP-1 orWISP-2 or WISP-3 showed a strong positive staining for collagen II. Thisdata indicated that WISP proteins promote the redifferentiation ofprimary porcine chondrocytes in culture.

Example 6 Articular Cartilage Explant Assay

An experiment was conducted to examine both the synthetic andprophylactic potential of WISP polypeptides on cartilage matrixturnover. This potential is determined by measuring matrix (i.eproteoglycan) synthesis and breakdown, as well as nitric oxideproduction, in articular cartilage. These parameters are evaluated inthe presence and absence of interleukin-1alpha. Articular cartilageexplants have several advantages over primary cells in culture. First,and perhaps most importantly, cells in explants remain embedded intissue architecture produced in vivo. Secondly, these explants arephenotypically stable for several weeks ex vivo, during which time theyare able to maintain tissue homeostasis. Finally, unlike primary cells,explants can be used to measure matrix breakdown. To set up cartilageexplants, articular cartilage must be dissected and minced which resultsin disruption of the collagen network and release of proteoglycans intothe culture media. This system thus mimics degenerative conditions suchas arthritis in which the matrix is progressively depleted.

The metacarpophalangeal joint of 4-6 month old female pigs wasaseptically dissected as described above. The cartilage was minced,washed and cultured in bulk for at least 24 hours at 37° C. and 5% CO₂in explant media, i.e. serum free (SF) LG DMEM/F12 media with 0.1% BSA,100 U/ml penicillin/streptomycin (Gibco), 2 mM L-Glutamine, 0.1 mMsodium pyruvate (Gibco), 20 μg/ml Gentamicin (Gibco) and 1.25 mg/LAmphotericin B. Articular cartilage was aliquoted into micronics tubes(approximately 55 mg per tube) and incubated for at least 24 hours inthe above media. Media was harvested and new media was added (alone orwith WISP polypeptides (IgG-fusion constructs) at various time points(0, 24, 48 and 72 hours).

Media was harvested at various time points and then assayed forproteoglycan content using the 1,9-dimethylmethylene blue (DMMB)colorimetric assay of Farndale and Buttle, Biochim. Biophys. Acta 883:173-177 (1985) as described above. PG release at 0 hours was used as abaseline measurement, and any samples with especially high or low PGrelease were discarded prior to treatment with WISP-1 polypeptide. Forall treatments, results represent the average of 5 independent samples.

At 48 hours after the first treatment, ³⁵S-sulfate was added tocartilage explants at a final concentration of 10 μCi/ml along withfresh media (with or without test compound). After an additional 12-17hours of incubation at 37° C., the media was removed and saved forsubsequent PG and nitric oxide (NO) analysis. The cartilage explantswere washed twice with explant media and digested overnight at 50° C. ina 900 mL reaction volume of 10 mM EDTA, 0.1M sodium phosphate and 1mg/ml proteinase K (Gibco BRL). The digestion reaction was mixed (2:1)with 10% w/v cetylpyridinium chloride (Sigma) to precipitate theproteoglycans and centrifuged at 1000×g for 15 minutes. The supernatantwas removed and formic acid (500 ml, Sigma) was added to dissolve thepellets. The samples were then transferred to vials containing 10 mlscintillation fluid (ICN) and read in a scintillation counter.

After 72 hours, the remaining articular cartilage explants were digestedas described above and assayed for proteoglycan content using the DMMBcolorimetric assay (referenced above).

When articular cartilage explants were treated with either WISP-3 (FIG.14) or WISP-1 (FIG. 15A), both basal and IL-1alpha induced cartilagematrix breakdown were decreased. In addition, WISP-1 inhibited bothbasal and IL-1alpha induced nitrix oxide production (FIG. 15B).

These results show that WISP polypeptides can protect against cartilagecatabolism. Given the fact that elevated levels of both nitric oxide andIL-1alpha are found in diseased joints, the ability of WISP polypeptidesto block activity of IL-1alpha and production of nitric oxide suggestthat WISP polypeptides can decrease the extent of tissue damage inarthritic joints.

Example 7 Transgenic Mice Expressing WISP-2

To test the effect of WISP polypeptides in vivo, transgenic mice werecreated which overexpress WISP-2 in their muscle by virtue of the myosinlight chain promoter. The transgenics were made using techniques knownin the art (Manipulating the Mouse Embryo: A Laboratory Manual,Beddington et al., Cold Spring Harbor Press, 1994; Transgenic AnimalTechnology: A Laboratory Handbook, Academic Press, New York, 1994). Thebones of these mice were examined at 14 weeks of age by standardhistology. Following sacrifice of animals, bones were fixed in 4%buffered formalin, followed by decalcification in Formical™ for 4-8hours. Samples were then processed for paraffin embedding and forhistological assessment. Three-micron thick step sections were cut andstained with hematoxylin and eosin.

As shown in FIG. 16, the hyaline cartilage compartments (i.e. the growthplate and the articular cartilage) appear to be expanded. These resultsare consistent with results presented in the Examples above showing theability of WISP polypeptides to induce cartilage cell differentiationand inhibit cartilage matrix breakdown. Thus, WISP polypeptides can havepotent effects on cartilage tissue in vivo. Treatment of an arthriticindividual with a polypeptide having such activity, namely one whichincreases the amount of cartilage, may prevent the disability and jointdestruction which can occur in arthritic patients.

1. A method of treating mammalian cartilage cells or mammalian cartilagetissue, comprising contacting mammalian cartilage cells or mammaliancartilage tissue damaged from a degenerative cartilagenous disorder withan effective amount of WISP polypeptide, wherein said WISP polypeptideis a polypeptide selected from the group consisting of: a) a WISP-1polypeptide comprising amino acids 23 to 367 of SEQ ID NO:3; b) a WISP-1polypeptide comprising amino acids 1 to 367 of SEQ ID NO:3; and c) aWISP-1 polypeptide having at least 95% identity to the polypeptide of a)or b), wherein said WISP-1 polypeptide stimulates chondrocyteproliferation or differentiation.
 2. The method of claim 1 wherein saidWISP-1 polypeptide comprises amino acids 23 to 367 of SEQ ID NO:3. 3.The method of claim 1 wherein said WISP-1 polypeptide is linked to oneor more polyethylene glycol molecules.
 4. The method of claim 1 whereinsaid WISP-1 polypeptide is linked to an epitope tag or immunoglobulinmolecule.
 5. The method of claim 1 wherein said cartilage cells ormammalian cartilage tissue is articular cartilage.
 6. The method ofclaim 1 wherein the degenerative cartilagenous disorder is rheumatoidarthritis or osteoarthritis.
 7. The method of claim 6 wherein thedegenerative cartilagenous disorder is rheumatoid arthritis.
 8. Themethod of claim 1 wherein the mammalian cartilage cells or mammaliancartilage tissue is contacted with the effective amount of the WISPpolypeptide in vivo.
 9. The method of claim 1 wherein the WISPpolypeptide is included in a pharmaceutically acceptable carrier.
 10. Amethod of treating mammalian cartilage cells or mammalian cartilagetissue, comprising contacting mammalian cartilage cells or mammaliancartilage tissue damaged from injury with an effective amount of WISPpolypeptide, wherein said WISP polypeptide is a polypeptide selectedfrom the group consisting of: a) a WISP-1 polypeptide comprising aminoacids 23 to 367 of SEQ ID NO:3; b) a WISP-1 polypeptide comprising aminoacids 1 to 367 of SEQ ID NO:3; and c) a WISP-1 polypeptide having atleast 95% identity to the polypeptide of a) or b), wherein said WISP-1polypeptide stimulates chondrocyte proliferation or differentiation. 11.The method of claim 10 wherein the injury is a microdamage or blunttrauma, a chondral fracture, an osteochondral fracture or damage tomeniscus, tendon or ligament.
 12. The method of claim 10 wherein saidWISP polypeptide is administered to a mammal by injection into saidcartilage cells or mammalian cartilage tissue or a joint of the mammal.13. The method of claim 10 wherein said WISP-1 polypeptide comprisesamino acids 23 to 367 of SEQ ID NO:3.
 14. The method of claim 10 whereinsaid WISP-1 polypeptide is linked to one or more polyethylene glycolmolecules.
 15. The method of claim 10 wherein said WISP-1 polypeptide islinked to an epitope tag or immunoglobulin molecule.
 16. The method ofclaim 10 wherein the mammalian cartilage cells or mammalian cartilagetissue is contacted with the effective amount of the WISP polypeptide invivo.
 17. The method of claim 16 wherein the WISP polypeptide isincluded in a pharmaceutically acceptable carrier.